CN103069010A - Diagnosis of viral infections by detection of genomic and infectious viral DNA by molecular combing - Google Patents

Abstract

A method for detecting in vitro the presence of a genome of a DNA virus or a viral derived DNA in an infected eukaryotic cell, tissue or biological fluid using Molecular Combing or other nucleic acid stretching methods together with probes, especially nucleic acid probes, having a special design. A method for monitoring in vitro the effects of anti-viral treatment by following the presence of genomic viral or viral derived DNA polynucleotides in a virus-infected cell, tissue or biological fluid. Detection of an infectious form of a virus using Molecular Combing and DNA hybridization. A kit comprising probes used to carry out these methods and a composition comprising the probes.

Description

By detected the diagnosis of the virus infection of genome and infectious virus DNA by molecular comb

[technical field]

It is easy to the present invention relates to use molecular comb and/or DNA to extend the specially designed probe of combination infectious virus or pathogenicity bo dna sequence dna, detects fast and accurately the method for the existence of in virus host cell, tissue or biological fluid that experimenter or patient obtain infectious virus DNA or other infectivities or pathogenicity bo dna sequence dna.To be tested for come external monitoring to resist-method of the effect of viral therapy by the existence of genomic viral polynucleotide in cell, tissue or the fluid of the patient's of virus infection virus-infections by following the tracks of.Detection based on the infectious virus of DNA hybridization.Comprise to implement method of the present invention probe test kit and comprise the composition of described probe.

[background technology]

[molecular comb]

The molecular comb technology has been disclosed in various patents and scientific publications, for example at US6, and 303,296, WO9818959, WO0073503, US2006/257910, US2004/033510, US6,130,044, US6,225,055, US6,054,327, WO2008/028931, WO2010/035140, and Michalet, Ekong et al.1997, Herrick, Michalet et al.2000, Herrick, Stanislawski et al.2000, Gad, Aurias et al.2001, Gad, Caux-Moncoutier et al.2002, Gad, Klinger et al.2002, Herrick, Jun et al.2002, Pasero, Bensimon et al.2002, Gad, Bieche et al.2003, Lebofsky and Bensimon2003, Herrick, Conti et al.2005, Lebofsky and Bensimon2005, Lebofsky, Heilig et al.2006, Patel, Arcangioli et al.2006, Rao, Conti et al.2007 and Schurra and Bensimon2009(see following quoted passage).Be described in these reference, belong to especially or be relevant to those technology of molecular comb, supply with and equipment publication cited above by reference merges at this.

The people such as Bensimon, US patent No.6,303,296, open DNA extends process, the people such as Lebofsky, the detection of the open genome sequence of WO2008/028931, reach the people such as Lebofsky, WO2010/035140A1, open based on the people the 4th of nucleic acid extension and molecular comb and the analytical procedure of the series connection of the D4Z4 on 10 karyomit(e)s repeat array.

Molecular comb is the direct visual technology that causes individual nucleic acid molecule, and be successfully used in the cervical cancer cell system (HPV) location of sequence of myc and particular person Papillomavirus (Papillomavirus), and on MYC/ papilloma virus (HPV18) amplicon that characterizes before being used for and special on its replication origin the research (Herrick of duplicating dynamics, et al., Cancer Res.65 (4): 1174-11792005).Nucleic acid in the tumour cell is reset relevant with the cancer result of this technology of use with amplification.The humans such as Herrick be specific to N-myc and c-myc gene probe and for detection of the HPV nucleic acid that contains total length HPV-18 and HPV-44 (～7.8kb) probe has been detected the tumor cell line IC1 that certainly cultivates, IC2, the nucleic acid that the quilt of IC4 and IC5 is combed.Conti, et al., Genes, Chromosomes ﹠amp; Cancer46:724-734(2007) studied the initial point activation of the HPV/MYC amplicon that contains specific HPV18 nucleotide sequence, and initial point activity and the crunode analyzed on the DNA that is extended by the molecular comb from the IC1 tumor cell line move.Conti focuses on the mechanism that relates to HPV and MYC nucleotide sequence and gene rearrangement is accredited as the sign of cancer cells with increasing.The tumour cell of cultivating has been used in these two researchs, and wherein the HPV nucleotide sequence is brought into play the effect of oncogene, but does not bring into play the effect of infectious gene papova nucleic acid.

By characterized the additive type that obtains from the Burkitt lymphomas clone of cultivating and the epstein-Barr virus DNA(Reisinger of integration, et al., Int.J.Cancer118:1603-1608(2006 by the FISH on the DNA that combs)).The purpose of Reisinger is the virus genomic genomic organization of research and understands and attempt the EBV of the form of additive type and integration in the eukaryotic cells that difference infects.Being extended from the DNA of the Burkitt lymphomas clone of cultivating and with covering gene group sequence by molecular comb, but not being terminal repetition son (TR) and inner repetition 1(IR1) the EBV-specificity DNA probing needle in district between the sequence detects.

The people such as the people such as Reisinger and Herrick do not describe the different color that uses with the rearrangement within this given zone that can generation can the allow identifying virus DNA-different hapten-marked probe groups of fluorescence array yet and detect the method for virus genomic rearrangement.This 2 reference is described cultured cells but is not the detection of viral DNA in tissue or the biological fluid.

On the contrary, in the present invention, the standard leaching process is modified to permission extracts viral DNA from virus particle, in order to analyzed the structure of viral DNA in the infectious particle by molecular comb.And the present invention has developed the method for extracting high-molecular-weight DNA from solid tissue such as cornea, and it is with compatible by the analysis of molecular comb, and permission detects the existence of the HSVDNA of infectious form in the cornea that infects.This 2 method never was described before the present invention.

Extending nucleic acid, especially virus or genomic dna provides the fixing nucleic acid of linear and parallel-chain, and preferred on suitable surface (for example glass slide on surface-process) with the elongation factor enforcement of controlling.After extending, it may hybridize the sequence-specific probe (Lebofsky, Heilig et al.2006) that can for example be detected by the fluorescence microscopy microscopy.Thus, particular sequence can be directly visual at single molecules level.The length of fluorescent signal and/or their number, and their intervals on slide glass provide directly reading of probe size and relative spacing.But, up to now, do not show that this technology can be used for detecting virus or virus sequence or cells infected or the tissue of the infectious form in the eukaryotic cells of infection or is present in the genome of the virus sequence of the sudden change of deriving in the Mammals fluid yet.

[virus structure and genome]

To cause human disease's virus based on the existence of coating, shell type, genome character (DNA or RNA, two strands or strand), size and target cell are divided into 7 groups of (International Committee on Taxonomy of Viruses, http://www.ictvonline.org; In last access on April 7th, 2011).The dna virus of parcel comprises poxvirus, simplexvirus and hepadnavirus.The group of non-parcel comprises adenovirus, papovavirus and parvovirus.

Can among viral species, see a large amount of kinds genome structure.Really, 1,000,000 kinds of dissimilar viruses (Breitbart and Rohwer2005) are arranged, although in them only about 5,000 kinds describe (Dimmock, Easton et al.2007) in detail.Viral genome is strand or two strands.The strand genome is comprised of unpaired nucleic acid, is similar to from 1/2 of the ladder of centre division.All RNA viruses is strand, except double-stranded reovirus.Double-stranded genome is comprised of the nucleic acid of 2 complementary pairings, is similar to ladder.All DNA virus is double-stranded, and except parvovirus, it has single stranded DNA.Some viruses are partially double stranded and genome (Collier and Oxford2006) the part strand such as belonging to those of Hepadnaviridae (Hepadnaviridae), containing.

Viral genome is ring-type, as in polyomavirus, or linear, as in adenovirus (Fields, Knipe et al.2007).Nucleic acid type is not relevant with the genome shape.Among RNA viruses, genome usually is divided into the separate part within the virus particle, and is called as segmentation.Each section a kind of albumen of usually encoding, and their are found in a housing together usually.For treating infective total precursor virus, every section is not required in identical virus particle, as showing (Collier and Oxford2006) by bromovirus.

For the virus with RNA or single stranded DNA, chain is called as justice (being called as normal chain) or negative justice (being called as minus strand), whether depends on itself and viral messenger RNA(mRNA) (mRNA) complementation (Fields, Knipe et al.2007).Positive sense viral rna is identical with virus mRNA, can be translated by host cell immediately thus.Negative-sense viral rna is complementary with mRNA, must be converted into just RNA by RNA polymerase before translation thus.The DNA nomenclature is to be similar to the RNA nomenclature, and wherein, the coding strand of virus mRNA is and its (-) complementation, and non--coding strand is the copy of its (+).Some single strand RNA viruses that are called as retrovirus (it comprises HIV) copy and produce DNA through enzymatic reversion record enzyme from its rna gene group in host cell.Then DNA is incorporated into host's genome by intergrase.Viral genome, or provirus remain in the genome of cell of infection, then as the partial replication of the DNA of host cell.

The genome size changes in the virus of different sorts or species greatly, the 309kb from the 1.7kb of hepatitis D virus (HDV) to poxvirus.RNA viruses is usually compared dna virus and is had more minigene packet size because when copying higher error rate, and have the overall dimension upper limit.Surpass this limit, the error when copying in the genome causes the useless or uncontested property of virus.For this is compensated, RNA viruses usually has the genome of segmentation, and wherein gene element is cleaved into more small molecules, reduces thus the chance of makeing mistakes.On the contrary, dna virus has larger genome usually, because the high frequency high fidelity of their replicative enzyme (Pressing and Reanney1984).

Virus can experience the heredity that occurs by different mechanism and change.The 1st process is called as genetic shift, and wherein the individual base mutation among DNA or the RNA becomes other bases.Most these point mutation are " silence " implications, and they do not change the albumen of genes encoding, but other can give evolutionary edge, such as the resistance of enantiopathy cytotoxic drug.Antigenic shift (Pan, Li et al.2007) occurs when in the genome of virus Main change being arranged.This can be the result of restructuring or reallocation.When this with influenza virus (Hampson and Mackenzie2006) occurs, can cause pandemic disease.RNA viruses usually exists as quasispecies or the group of the virus of identical species, but slightly different genome nucleotide sequences is arranged.This quasispecies is the main target (Metzner2006) of natural selection.Secondly, the genome of segmentation is also given evolutionary edge; Have the not homophyletic of the genomic virus of segmentation can reorganization and combination gene and generation have the progeny virus of unique and new feature.This is called as reallocation or viral sexual intercourse (Goudsmit1998).Finally, the genetic recombination of process that then the DNA splitting of chain joins the end of different dna moleculars to can appear.This occurs in the time of can working as viral simultaneously cells infected, and the restructuring that studies show that of virus evolution spreads (Worobey and Holmes1999) in the species of research.Restructuring is common (Umene1999 for RNA and dna virus; Lukashev2005).

HSV is among the larger virus of diameter 150nm～200nm.They are encapsulated in the coating that contains the glycoprotein furcella interior (Wildy, Russell et al.1960) of loosely assembling.Such as the virus of other parcels, simplexvirus tends to by organic solvent or stain remover deactivation, and unstable outside host's health.In some viruses, the icosahedron capsid cage firmly twines the core of the double-stranded DNA of albumen spindle body.The genome sequence of the HSV-1 strain 17 of publishing first in 1988 is listed in each chain and contains 152,261 residues (Genbank accession number No.:NC_001806.1; Last access on April 7th, 2011) (McGeoch, Dalrymple et al.1988).The HSV genome is considered to the section by 2 covalency joints, long (L) and short (S) district's composition (Roizman1979).The L district is by by a pair of relatively unique sequences (U of directed duplicon element side joint _L) form and (to be referred to as R _L, terminal and internal copy is referred to as TR especially _LAnd IR _L) (Wadsworth, Jacob et al.1975).The S district is similarly by IR _S, U _SAnd TR _SForm.R _LAnd R _SThe sequence of element is different, except at the genome end 400bp(bp being arranged) direct repeat, be referred to as sequence, and at least one further copy see the connection (Roizman1979 in L and S district with the orientation opposite with the end copy; Davison and Wilkie1981).Each end has the residue that overhangs, 3 '-hydroxyl dissociate (Mocarski and Roizman1982).HSV DNA prepared product contains 4 kinds of sequences of equimolar amount-orientation isomer, wherein U _LAnd U _SEach is independently to place (Hayward, Jacob et al.1975 about one of the 2 kinds of possible orientations of the junction between L and the S sequence; Bataille and Epstein1997).A kind of isomer is defined as prototype (Roizman1979).HSV-1DNA has the based composition (McGeoch, Dalrymple et al.1988) of 68.3%G+C.It is not constant that G+C content runs through genome; 6.6kbp R _SElement departs from the most significantly from mean value, and 79.5% G+C content (McGeoch, Dolan et al.1986) is arranged.The genome of HSV-2 is fully order-checking not, and it is the genome of simulation HSV-1 closely, although a little more G+C rich is arranged.

The genome polynucleotide sequence of multiple virus is well known to those skilled in the art and merges by reference, reference has Fields, B.N., D.M.Knipe, et al. (2007), Fields'Virology.Philadelphia, Wolters kluwer/Lippincott Williams ﹠amp; Wilkins and can passing through Http:// www.ncbi.nlm.nih.gov/genomes/genlist.cgi taxid=10239﹠amp; Type= 5﹠amp; Name=VirusesThe NCBI viral genome database of access (last access on April 7th, 2011).

[viral pathogenesis]

Identified and permitted eurypalynous viral pathogen.For the recently review to virus infection, can mention the reference of the people (Fields, Knipe et al.2007) such as Fields and this article, it merges by reference.When virus by as breathe the air mix virus, the process of the food that food mixes, or virus infection occurs by contacting with the having property of people that infects virus when entering health.Virus infection also can be caused by sting.In virus infection, the cell of virus attack health is interior so that breeding.Then virus expand to other cells, and repeat this process.This process of virus infection causes characterizing vicissitudinous various symptom, and seriousness depends on type and the individual factors of virus infection.The common symptom of virus infection comprises tired, influenza-sample symptom and heating.

Being permitted eurypalynous virus infection, such as common cold, is self limiting in common Healthy People.This expression virus infection causes the disease certain hour, then its dissolving and transference cure.But some are in the risk of the serious complication of development virus infection.In addition, the virus infection of particular type, such as HIV/AIDS, be not self limiting and cause serious complication and/or can finally cause death.As an example of serious damage, the viral infection of measles of some forms causes the inflammation of brain, and thereby permanent brain injury.

The eurypalynous virus of being permitted that causes virus infection widely or virus disease is arranged.For example, there be different the causing above 200 kinds to catch a cold or the virus of upper respiratory tract infection.Other common viruses comprise the influenza virus that causes influenza or influenza.Epstein-Barr virus and cytomegalovirus cause infectious mononucleosis, and varicella zoster virus (VZV) causes zoster and chicken pox, and HIV causes AIDS.

Virus has different mechanism, and they produce disease in biology thus, and it depends on viral species widely.Mechanism at cell levels mainly comprises lysis, breaks and subsequently necrocytosis.In multicellular organism, if enough necrocytosiss, full biology can begin to suffer effect.Although the destruction that virus causes healthy stable state causes disease, they also can relatively harmlessly exist in biology.One regular meeting comprises that the hsv (HSV) that causes cold blister keeps the ability of dormant state within the person.This is called as latent period (Margolis, Elfman et al.2007), and is the feature of whole simplexviruss, comprises the EBV that causes the gland heating, and VZV.Majority have infected at least a (Whitley and the Roizman2001) among the HSV of these types.But these latent viruss can be useful sometimes, can increase the directed toward bacteria pathogenic agent such as the existence of virus, such as the immunity (Barton, White et al.2007) of yersinia pestis (Yersinia pestis).On the other hand, the chicken pox of hiding infects to be returned in the life subsequently, as is called as the disease of zoster.

Some viruses can cause throughout one's life or chronic infection, although host's defense mechanism wherein, virus continues to copy (Bertoletti and Gehring2007) in health.This is common in hepatitis B and infection with hepatitis C virus.The people who infects chronically is known as carrier, because they are as the carrier of infectious virus.In having the group of a high proportion of carrier, disease is known as endemic (Rodrigues, Deshmukh et al.2001).Opposite with acute cracking performance virus infection, this retains the compatible interaction of hint and host living beings.

It is an example of virus infection that HSV-1 infects.HSV produces the trend name (Taylor, Brockman et al.2002) that spreads rash with some herpes infections.It is the popular name of extended familys, and its member comprises: herpes simplex 1 and 2, the origin cause of formation that heating blister and reproduction are infected; VZV, the origin cause of formation of chicken pox and zoster; Cytomegalovirus (CMV), it affects sialisterium and other internal organ; EBV, related with the infection of lymphoid tissue; And some viruses (HSV-6 ,-7 and-8) of identifying recently.The significant feature of family is that it is to the trend of the infection of Virus latency and recurrence.

HSV infects, and usually generically is called as bleb, usually the target mucous membrane.Then crack or the otch on cell entry film surface double near the substrate immediately and the epithelial cell.This causes inflammation, oedema, lysis, and characteristic thin-formation of the vesica of wall.The principal disease of HSV is face simplex (oral cavity, eye and pharynx), reproduction bleb, newborn infant's bleb and the disease of sending out.The herpes labialis that is called as in addition heating blister or cold blister is that the HSV-1 of modal recurrence infects.Vesica is usually in the mucocutaneous junction of lip or long at adjacent skin.Herpes keratitis (being also referred to as herpes ophthalmicus) is the infection inflammation of eye, and wherein latent virus enters trifacial ramus ophthalmicus but not ramus of mandible.The sand sample sensation that preliminary symptom is eye, conjunctivitis, sharp pain, and to photaesthesia.Some patients also develop characteristics branch or opaque corneal injury.In 25%～50% situation, keratitis be recurrence with chronic and can disturb vision.

In more than 90% situation, primary infection, asymptomatic (Liesegang1989) occurs in oral mucosa, as the result who contacts with the particle of the infection of saliva.Soon, the cyclisation of linear viral genome and dna replication dna are at initial point initial (as summary: Boehmer and Lehman1997) after infecting.Originally dna replication dna is undertaken by θ mechanism, switches to subsequently σ or rolls ring mode, to produce long head-to-tail concatemer.From many dna replication dnas forks that homologous recombination occurs, the genome isomerization of sequence-mediation reaches other events, causes the formation of network of expansion of the DNA intermediate of branch.Finally, these structures are dissolved into the unit-the length mrna group, and packing is advanced pre-assembled housing.But, can be within nucleocapsid than the short dna molecular of total length standard HSV-1 viral DNA shell, as long as they contain cutting/packaging signal (Vlazny, Kwong et al.1982).But, significantly be shorter than the genomic housing of the genomic HSV-1 of containing of standard virus and do not infect.

After copying in epithelium, it is neurone that virus increased before hiding.1 type HSV mainly enters trident, or the 5th cranium, nerve, and it has the innervation of expansion in dental sector, and 2 type HSV hide in the neuroganglion that waist-sacrum spinal nerves is done usually.By various priming factorses such as heating, the UV radiation, stress or physical abuse, virus is moved to body surface and is produced local skin or membrane damage, usually with infect before identical site.The master site of HSV-1 is gasserian ganglion owing to hide, and is responsible for the sensory nerve domination of face, and most recurrences are positioned at eye or lip.The serum Flow Behavior 24.5%～67% of HSV-1 in general group has the herpes labialis (Liesegang2001) of 30～70% positive subjects experience recurrence.Really, almost everyone can present HSV-1 infection event potentially, because to recently studies show that of use polymerase chain reaction (PCR) of tissue after death, almost 100% at least 60 years old people exists in gasserian ganglion and has several HSV-1 genomes copies (Wang, Lau et al.2005).Eye, and special cornea is the 2nd position frequently that HSV-1 infects.The prevalence rate of herpes keratitis is 149/100,000(Liesegang, Melton et al.1989), annual more than 30 new events/100,000 occupants (Labetoulle, Auquier et al.2005).In case eye is by the herpes infection clinical impact, the recurrence speed in 2 years of following the tracks of is that the recurrence speed in 23% and 5 year is 40%(Wilhelmus, Coster et al.1981; Liesegang1989).Vision prognosis is poor, especially in degree of depth corneal infection (interstitial keratitis), and the eye that reaches 60% impact is less than 20/40 visual acuity (Kabra, Lalitha et al.2006) after following the tracks of 5 years.As a result, be the leading reason of losing one's sight at whole world HSV-1.In less developed country, the HSV-1 eye infections accounts for about 10%(Pramod of the patient of nursing cornea clinic, Rajendran et al.1999), although and used part or oral cavity antiviral agent at nearest 30 years, herpes simplex keratitis also remains the most frequently origin cause of formation (group 1998) of the infectious corneal opacity in the most developed area in the world, account for about 10% patient experience corneal transplantation (Leger, Larroque et al.2001).And, the natural risk that HSV-1 brings back to life in the cornea of transplanting recently after operation the 1st year be about 25%(Lomholt, Baggesen et al.1995), and about 33% primary graft failure (removing without graft) has shown and has been relevant to that HSV-1 is genomic in the cornea exists (Cockerham, Bijwaard et al.2000).These data interpretations why HSV-1 remain the major cause of corneal transplantation failure, account for the top and bottom transplant again about 22%.

The clinical complication of the infection of latent period and recurrence with advancing age, cancer chemotherapeutics or relax other state of an illness of immune defense and become more serious.HSV therein the most common and in AIDS patient serious conditioned pathogen (Ramaswamy and Geretti2007).Almost this group of 95% can experience by these viral skin mucous membrane, the repeatedly outbreak of intestines and ophthalmic.

Human immunodeficiency virus (HIV) causes acquired immune deficiency syndrome (AIDS) (AIDS) (Weiss1993; Douek, Roederer et al.2009), immunity system begins to lose efficacy the slow virus (member of retrovirus family) that causes the state of an illness of life-threat opportunistic infection among the people.HIV infects by blood, seminal fluid, and vaginal secretions, the transfer of front ejaculation secretory product or breast milk occurs.Within these body fluid, HIV exists as the virus within the immunocyte of free virus particle and infection.4 kinds of main routes of transmission are unsafe sexual intercourses, the pin of pollution, and the mother of breast milk and self-infection is to the baby's of its birth propagation (vertical transmission).Developed country to blood product in the screening of HIV eliminated widely propagation by blood transfusion or the blood product that infects.

HIV among the people infects and is thought prevailing disease by world health organization (WHO).Comfortable 1981 its be found to 2006, AIDS and kill more than 2,005 million peoples.HIV infects about 0.6% world population.Only in 2005, AIDS asked for and has estimated 2.4～3.3 hundred ten thousand life, wherein more than 570,000th, and children.Anti-retroviral agent treatment reduces mortality ratio and the morbid state that HIV infects.

Viable cell in the HIV main infection human immune system (specificly is CD4 such as helper T cell ⁺The T cell), scavenger cell and dendritic cell.HIV infects and causes low-level CD4 by 3 kinds of dominant mechanisms ⁺The T cell: the 1st, the direct virus of the cell of infection is killed; The 2nd, the apoptosis speed that increases in the cell of infection; And the 3rd, the CD4 of infection ⁺The cd8 cell toxicity lymphocyte that the T cell is identified the cell of infection kills.Work as CD4 ⁺The T cell count is reduced to critical level when following, cell-mediated immunity forfeiture, and health becomes progressively and more is sensitive to opportunistic infection.

Untreated majority is finally developed AIDS by the people that HIV-1 infects, and finally dies from opportunistic infection or pernicious (Lawn2004) related with immune carrying out property destruction.HIV is with quilt virus, and the variable velocity of host and such environmental effects develops into AIDS; Most can the infection within 10 years at HIV developed into AIDS: some can make progress more faster, and some can consume the more much longer time (Buchbinder, Katz et al.1994; 2000).Increase the people's who is infected by HIV predicted life with the treatment of anti--retrovirus medicine, even after the HIV progress is for diagnosable AIDS, with the mean survival time of anti-retroviral treatment as being estimated as in 2005 greater than 5 years (Schneider, Gange et al.2005).Without the anti-retroviral treatment, some suffers from the generally death (Morgan, Mahe et al.2002) in 1 year of people of AIDS.

[diagnosis of virus infection and treatment]

Virus disease is by various clinical and laboratory method diagnosis.These comprise: the clinical evaluation of symptom, and virus in cell or the animal culture is separated, and to the serology test for the antibody of virus.The diagnosis of carrying out virus infection starts from gets sufficient individual and family's medical history, comprises symptom, and finishes health check-up.Diagnose some virus infectiones, such as seasonal influenza, can carry out based on historical and health.The blood test can be carried out such as complete blood counting.The blood counting is measured dissimilar hemocytes fully, comprises white corpuscle (WBC) number.Dissimilar WBC numbers are in infectious process, such as increasing in the characteristic mode during the virus infection.Cultivating test also can carry out.This test needs to be got small sample from suspecting by the body region of virus infection, and at laboratory growth sample, causes the microorganism type of disease with mensuration.Common sample comprises from larynx with culture, blood and from those tests of the phlegm of lung.Diagnostic test also can comprise and waist puncture be also referred to as thecal puncture, and it relates to pin and extracts cerebrospinal fluid (CSF) small sample from backbone.The white corpuscle of test CSF sample and can be in backbone or brain other virus infection indexs, such as viral meningitis.Can implement the X-ray, with the diagnosis of more auxiliary virus infectiones.This can comprise the chest X-ray of the situation of the virus bronchopneumonia of getting suspection.Can carry out other test, but so that eliminating or affirmation associated virus infect or cause the other diseases of similar symptom, infect such as the Secondary cases bacterium.

HSV infects by various clinical and laboratory method diagnosis, comprises the clinical evaluation of symptom, separates in cell or animal culture, reaches the serology test for the antibody of HSV.Mouthful or phallic mucous membrane on little, pain, vesiculation damage, lymphadenopathy and exudate are the typical diagnostic symptoms of herpes simplex.Further diagnosis support is use Giemsa by checking from this, and Wright(is also referred to as the Tzanck preparation), or Papanicolaou(Pap) base portion of damage of method dyeing scrapes and consider to be worth doing and available.Multinucleate cell, the existence of acidophilia occlusion body can be assisted and be set up herpes infection in giant cells and the nuclear.But this method can not distinguished HSV-1, HSV-2 or other simplexviruss, and it needs more specific inferior somatotype.And laboratory culture and fc-specific test FC are immunosuppressant and suffer from seriously for diagnosis, and the neonatal patient of the herpes infection of sending out is necessary.To organize or fluid sample imports primary cell line such as monkey kidney or human embryo kidney tissue culture, then observe the cytopathic effect within 24～48 hours.Sample or cell culture are used the direct test of fluorescence antibody or use specific probe or can distinguish HSV-1 by the DNA detection of polymerase chain reaction (PCR) amplification, HSV-2 and the HSV that is closely related, but be not to be complete or infective useful instrument for the described viral genome of confirming to see sample.Although serological analysis is useful for primary infection, its disease for recurrence is inc, because the antibody titers to HSV does not increase usually behind infection and recurrence.

Several doses is available for the HSV treatment of infection.Acyclovir is the most effective treatment of developing so far, and it is that non-toxicity and high special are in HSV.Famciclovir and valacyclovir are alternative medicine.Be applied to duration and the minimizing virus shedding of the topical medications cutting infection of reproduction and injury of mouth.Systemic treatment is available for more serious complication such as herpes keratitis and the bleb sent out.Fresh evidence indication, dosage every day of oral cavity acyclovir continue 6 months～1 year period can be in the reproduction bleb of prevention of recurrence effectively.Contain menthol, the cold blister pharmacological agent of the OTC (over-the-counter) of camphor and local anesthetic reduces pain and avoids the infection of Secondary cases bacterium with protecting, but they probably do not affect the development of virus infection.Some protections in suppressing cold blister can be by getting amino acid lysine at the very early time per os of recurrence and obtaining.But current treatment does not reduce the load of DNA matrix, can not reduce thus the risk of further virus rescue.Ideally, the last weapon that infects for HSV-1 should be to be present in durably in the cell, avoids sensitivity problem to reach, and if possible, reduces virus genomic load.The new anti-virus agent treatment that is intended to be eliminated by the effect of endonuclease viral DNA is under development.

From the tear (Fukuda from healthy and ill patient, Deai et al.2008), cornea is scraped bits (El-Aal, El Sayed et al.2006) and cornea explant button (Crouse, Pflugfelder et al.1990) method (Llorente, the Hidalgo et al.1998 of use PCR-based; Gonzalez-Villasenor1999; Kessler, Muhlbauer et al.2000; O'Neill, Wyatt et al.2003; Kimura, Ihira et al.2005; Namvar, Olofsson et al.2005; Strick and Wald2006; Susloparov, Susloparov et al.2006; Engelmann, Petzold et al.2008; Sugita, Shimizu et al.2008; Tanaka, Kogawa et al.2009; Wada, Mizoguchi et al.2009; Yu, Shi et al.2009) or to DNA/DNA hybridization (Nago, the Hayashi et al.1988 of the cell that infects; Kotronias and Kapranos1998) viral DNA of implementing HSV in the cornea detects.But, the genomic detection of infectious HSV and character in none cell that allows in single the analysis, infecting of these methods.

HSV is one type virus disease, to this, the widely technology that is used for diagnosis HSV infection has been disclosed in various patents and publication, EP0139416 for example, EP0263025, WO0202131, WO2004036185 and Matsumoto, Yamada et al.1992, Kotronias and Kapranos1998, Kessler, Muhlbauer et al.2000, Namvar, Olofsson et al.2005, Susloparov et al.2006, Sugita, Shimizu et al.2008, and Yu, Shi et al.2009.

The most reliable method of diagnosis HSV infectious diseases is to separate to be used for the virus determined, but this method needs culturing cell and needs several days with definite.Immunological method is also arranged, but they are normally insecure, and difficult enforcement, especially during latent period.The method of having developed a large amount of PCR-based is used for comparing by the sensitivity of the possible enhancing of conventional means with faster to time of result, but these methods do not allow to analyze full HSV genome, and the unpredictable infectivity that contains the sample of viral nucleotide sequences is not because the complete genome of described virus is tested.In situ hybridization (Nago, Hayashi et al.1988 have been reported in; Kotronias and Kapranos1998) or dot blotting DNA-DNA hybridization (Matsumoto, Yamada et al.1992) in the HSV genome detects in cell direct method, but these methods can not be measured the type of HSV.The quick of HSV disease arranged, and responsive and specific diagnosis is urgent example.Therefore clinical demand to the quick and responsive instrument of developing auxiliary HSV diagnosis is arranged.Also maintain the demand of the instrument of the somatotype that infects for HSV.The Rapid identification of the specific pathogen that relates in the virus infection provides the information of determining suitable treatment within the short period of time that is used in.

Carry out the genomic conformational analysis of HSV-1 behind cracking performance or the latent infection by the people's such as Gardella gel electrophoresis system (Gardella, Medveczky et al.1984).In these gels, compare the linear forms of answering, ring molecule presents more harmonic motion characteristically, and this has allowed the detection of the hsv gene group of hiding of additive type form.But this technology is not distinguished the genomic isomer of HSV in the cell or tissue of infection.Other technologies such as pulsed field gel electrophoresis (PFGE) have been used for providing see clearly (Severini, the Morgan et al.1994) to the mechanism of HSV dna replication dna.This test family is highly consuming time, and needs southern blotting technique in the top and bottom, hint hot operation, long migration and/or time shutter.

Another virus of having reported multiple determination and analysis method is the human immunodeficiency virus, or HIV, comprises HIV-1 and HIV-2.HIV-1 carrying capacity in the blood plasma as being measured by the copy number of HIV-1RNA, is widely used main laboratory marker in clinical practice.Higher virus load is directly related in the individuality that HIV-1-infects more rapid progress to AIDS.The validity of highly activated anti-retroviral treatment (HAART) is also evaluated by HIV-1 carrying capacity in the measurement blood plasma.The patient is considered to successfully be treated by HAART, and below the HIV-1 carrying capacity in the blood plasma rested on the commercial detectability of measuring, it was the HIV-1RNA/ml blood plasma of 50 copies at present.But although its clinical success, HAART can not eradicate virus, comprises the static CD4 of latent infection mainly due to various viral storages ⁺Cell retain (Hermankova, Siliciano et al.2003; Siliciano, Kajdas et al.2003).Recently research shows, virus replication and evolving continues in some patients, even the HIV-1RNA in blood plasma is not detectable, and successful (Frenkel, Wang et al.2003 are thought in treatment in addition; Havlir, Strain et al.2003; Ramratnam, Ribeiro et al.2004; Chun, Nickle et al.2005; Tobin, Learn et al.2005).HAART as the result of the exploitation of the HIV-1 strain of drug resistance unsuccessfully is common problem (del Rio2006).Thus, specific attention should give to characterize residual the copying of HIV-1 by its molecular marked compound in the research peripheral blood lymphocytes (PBMC).Especially, the quantitative amount of the HIV-1RNA of cell-association and proviral DNA.In these, the amount of proviral DNA can reflect the size of the cell bank of latent infection.But, the systematic Study of the relation between cell HIV-1RNA/DNA level and the treatment result by among the PBMC under HAART extremely the HIV-1RNA/DNA of low copy number hinder.Therefore, the exploitation of quantitative extremely sensitive method that is used for the HIV-1RNA/DNA of cells form is necessary.

In real time reverse transcription-PCR(RT-PCR) is preferred method (Douek, the Brenchley et al.2002 that is used at present quantitative cell HIV-1RNA/DNA; Fischer, Joos et al.2004).But, although their tolerance range and specificity, use TaqMan detect the list of chemistry-step real-time RT-PCR method can not be in the sight of total cell RNA/DNA every reaction quantitative＜100 HIV-1RNA/DNA target (Espy, Uhl et al.2006) of copy reliably.This cause to produce the possibility of vacation-negative findings, when research is from patient's PBMC material under HAART, especially when the clinical material of the amount of restriction when being available for analysis.Use can be more responsive (Espy, Uhl et al.2006) based on the green detection chemistry of SYBR with the method that detects HIV-1RNA/DNA, but tends to vacation-positive findings, because dna binding dye is not with sequence-specificity mode combination.Along with the theoretical detectability of a molecule of every reaction, sleeve type PCR thinks to compare more sensitive method of PCR in real time.But, can produce only half-quantitative data with this method.In addition, it needs manpower-experimentation intensive and consuming time.On the contrary, as described below, the contriver has found that molecular comb allow to detect unique event, and can be used as detect and the cell of quantitatively patient's infection in the extremely sensitive method of HIV proviral DNA.

For judging viral infection, the use that Detection of antigen is measured is increasing, especially based on those of monoclonal antibody.Use is for the quick detection of nucleic acids of the specific probe of DNA or RNA, or amplification method is other options for several viruses.These have advantage, and its sensitivity like this can conceivably detect viral nucleic acid to them in single cell that infects.For example, permission by additive method usually the round pcr of the enzymatic amplification of the DNA of undetectable small quantity be widely used in and detected several viral agents such as HSV(Rowley, Whitley et al.1990), human immunodeficiency virus (HIV) (Ou, Kwok et al.1988) and the genomic part of human papillomavirus (HPV) (Shibata, Arnheim et al.1988).

The remarkable restriction of PCR is its integrity that does not allow to confirm the complete genome that detects, thereby not can be the genomic signature criteria of complete infectious virus of finding in the sample of test.PCR also lacks testing needle thus to the specificity of the efficient needs of the antiviral agent treatment of infectious virus particle, because its result does not indicate whether that the viral polynucleotide that detect are infective.Serological method is direct-detection infectious virus polynucleotide not, and such as the virus genom DNA of karyomit(e)-integration, and the virus antigen that needs the experimenter to produce before detecting for the immunne response of virus or enough amount is present in the sample.

[viral oncogene detect and by virus former-the oncogene activation]

Many cancers are derived from virus infection; This animal such as bird, but also especially genuine in the people.The world, the international cancer of WHO research institution estimates that the people's cancer at 2002 20% is caused by infection, wherein 10～15% cause (Carrillo-Infante, Abbadessa et al.2007) by one of 7 kinds of different virus.But only the small portion human or animal can develop into cancer after infecting.Tumour virus has various forms: have the genomic virus of DNA, such as the HPV(cervical cancer), hepatitis B virus (liver cancer), and EBV(one class lymphoma), and have the virus of rna gene group, (HCV) can cause cancer such as hepatitis C virus, as have the retrovirus of DNA and rna gene group can (people T-parent's lymph venereal disease poison and hepatitis B virus, its two and single-stranded DNA viruses normal replication as mixing copies component but also have retrovirus).

Directly oncovirus mechanism relates to already present oncogene (former-oncogene) in other Viral Carcinogenesis gene Insertion Into Host Cell (acutely-transforming virus) or the enhancing gene group (slowly-transforming virus) (as summary, (Parsonnet1999)).Acutely-transforming virus in, virus particle is carried the gene of coding over-activity oncogene, it is called as virus-oncogene (v-onc), and just v-onc expresses, and just transforms the cell that infects.On the contrary, slowly-transforming virus in, insert viral genome, especially because viral genome is inserted is the obligate part of retrovirus, near in the host genome former-oncogene.Viral promotors or other transcription regulatory elements, and then, cause cross expressing of this former-oncogene, itself so that induce not controlled cell proliferation.Because it is not to be specific to former-oncogene that viral genome is inserted, and can be low near the insertion machine of this former-oncogene, slowly-transforming virus compares acutely-transforming virus has very bearing tumor latent period, and it has carried virus-oncogene.

When their cells infecteds and be continuously ring-type episome or plasmid, when copying independently with host cell DNA, some viruses are tumorigenesis (epstein-Barr viruses and Kaposi sarcoma-related simplexvirus).Also there is indirect Viral Carcinogenesis and relates to and infect the chronic nonspecific inflammation that more than ten year occurs, in the situation such as the liver cancer of inducing at HCV-.These 2 mechanism are different on their biology and epidemiology: directly tumour virus must have at least one virus copy in the tumour cell of at least a albumen of every expression or RNA, and it is carcinous that it causes cell to become.Exogenous virus antigen is expressed in these tumours, thereby the people of immunosuppression such as AIDS or transplant patient are in the risk of the cancer of higher these types.

[based on the gene or the cell therapy that use virus vector]

Gene therapy relates to inserts individual cell and tissue with gene, and with the treatment disease, such as heredopathia, wherein harmful mutation allele of gene is had the replacement of function.Although described technology is still in its infancy, it has used and has had some successful.Virus vector is usually to use this genetic material is sent into the instrument of tissue (gene therapy) or cell (cell therapy) on the science.These virus vector main sources are from slow virus, retrovirus, adenovirus or simplexvirus (as summary, (Thoma, Ehrhardt et al.2003)).

Genetic material in slow virus or the retrovirus is the RNA molecular form, and their host's genetic material is dna form.The genome of these viroids can be modified by target gene sequence to be transferred being inserted in the tissue that infects or the cell.As wild-type virus, recombinant virus can with some enzymes, namely ThermoScript II and intergrase are together with its RNA transfered cell.This must produce the DNA copy from its RNA molecule from the RNA of recombinant virus molecule before it can be incorporated into the genetic material of host cell.After this DNA copy produced and be free in host cell nuclear, it must be incorporated into the genome of host cell.That is, it must be inserted by another enzyme (being called as intergrase) that virus is carried the karyomit(e) in the cell.If this host cell divides subsequently, its offspring can all be contained new gene.

Can be that intergrase can insert the genetic material of virus any optional position in the host genome from one of problem of the gene therapy of the use of slow virus or retrovirus; It clamp-ons karyomit(e) at random with genetic material.If in one of protogene of the lucky Insertion Into Host Cell of genetic material, this gene can destroy (insertion mutagenesis).Just for the gene of regulating cell division, not controlled cell fission (that is, cancer) can occur such as fruit gene.This problem begins recently by utilizing strand target-seeking endonuclease (Grizot, Smith et al.2009) or by comprising that particular sequence imports the control region, seat (Zhou, Zhao et al.2007) of specific chromosomal foci with integration site and to be determined such as being used for.

Adenovirus is the virus of carrying their genetic material with the double-stranded DNA form.They cause the respiratory tract among the people, intestines and eye infections (especially common cold).When these virus infection host cells, they import the host with their dna molecular.The genetic material of adenovirus does not merge to the genetic material of host cell.Dna molecular keeps being free on host cell nuclear, and the instruction in this extra dna molecular is just like any other genetic transcription.Unique difference is that these extra genes do not copy when cell is about to experience cell fission, and the offspring of this cell can not have extra gene thus.Adeno associated virus (AAV) from parvovirus family is to have the genomic small virus of single stranded DNA.Wild-type AAV can be on the 19th karyomit(e) specific site to insert genetic material (Huser and Heilbronn2003) near 100% degree of certainty.But restructuring AAV, it does not contain any virogene and therapeutic genes only, and unconformability arrives genome.As a result, need to use again in the cell mass of growth with adenovirus carrier or AAV treatment meeting, although be incorporated into the development (Douglas2007) that the genomic disappearance of host cell should give protection against cancer in advance.

Simplexvirus is at present as gene transfer vector and since their lid the specific advantages of other virus vector.The very high transgenosis capacity that also is virus particle among the specific characteristic of the carrier that HSV originates allows to carry long exogenous DNA array, virus genomic hereditary complicacy, allow to produce the carrier of many dissimilar decay with oncolytic activity, and hsv vector invasion and attack are from feeling ganglionic neurone and set up therein the throughout one's life ability of non--toxicity latent infection, and transgenosis can be strongly and long-term expression therein.3 kinds of inhomogeneity carriers can come from HSV: the carrier of the decay of replication is arranged, without the auxiliary dependent form carrier of recombinant vectors and the defective that is called as amplicon of replication.The replication defect type hsv vector is by immediately one or more-early gene, for example disappearance manufacturing of ICP4, and then it provided with trans by the clone of complementation.The promise therapeutical agent that molten cancer hsv vector is cancer.Should based on the carrier of HSV in neurospongioma, test in melanoma and the ovarian cancer patients.

The contriver has been surprised to find that the molecular comb and the elongation technology that make up with specially designed probe or probe groups can be used for utilizing molecular comb and/or the diagnosis of DNA elongation technology, detect or monitor the experimenter who carries infectious virus DNA and other pathogenicity bo genes.These diagnostic are applied in usually to unknown in the prior art of the molecular comb that the DNA sample that the cultured cells system of knowing obtains carries out easily.Although molecular comb is applied to chromosome analysis before, do not recognize applicable other pathogenicity bo polynucleotide that can usefully be applied in judging viral infection or the test sample of this method or this method before it.On the other hand, the contriver has been surprised to find that directly can be activated by what detect molecular comb process and these samples from the DNA of the certainly experimenter's of virus-infection biological sample acquisition, infectious virus DNA evaluation.These methods overcome the many problems related with existing Diagnosis of Viral Infections method, and are provided for detecting and monitor among the experimenter of virus-infection or the patient infectious virus polynucleotide easily, quick and accurate method.

[summary of the invention]

The invention provides reliably, simply, fast and inexpensive way is to use in experimenter or patient's sample and can be with at least 5%, 10%, the specific probe of 25%, 50%, 75% infectivity or the combination of pathogenicity bo polynucleotide sequence, molecular comb and/or the DNA elongation technology of the probe groups combination of especially being combined with 80～100% infectivity or pathogenicity bo polynucleotide sequence detect infectivity or pathogenicity bo polynucleotide sequence, comprise virus genome sequence.Method of the present invention is external carries out.

The contriver has found that molecular comb detects viral DNA or the viral source-DNA that reaches in mammalian cell, tissue or the biological fluid that quantitatively infects for (i), such as the infectious herpes simplex virus dna in the cornea that infects, (ii) follow the tracks of the virus replication and the viral genome that in the mammalian cell tissue that infects or biological fluid, occur and reset, and identify that (iii) infectious virus polynucleotide or infectious virus polynucleotide in cell, tissue or the biological fluid that is present in infection are strong technology.

Be unlike in the prior art that detects virus in the cell or tissue of infection, molecular comb technology of the present invention can be carried out simultaneously, and does not need process how consuming time and costliness.The diagnostic tool that method of the present invention is polluted or infected as the virus that detects in the cell or tissue is applicable easily, for useful to the effect of measuring antiviral agent in the effect of the amount of the mammalian cell that infects or infected in tissues venereal disease poison or infectious virus polynucleotide or virus replication or effect and treating by quantitatively or in addition estimating this treatment.The contriver has found that molecular comb has overcome the past method with detection or judging viral infection, comprises many problems of the method association of PCR-based, and provides easily and immediate mode.Particular implementation of the present invention comprises following embodiment.

Detection of biological imitate viral genome in the product or the method for infectious virus polynucleotide, it comprises from described sample separation, extraction or obtains in addition polynucleotide; The described polynucleotide of molecular comb are to form the polynucleotide that extend; The polynucleotide of extension are contacted with the probe of one or more identification infectious virus or genomic viral polynucleotide sequence; The hybridization of the sample that detection probes and quilt are combed.Polynucleotide can be infectious gene papova DNA or infectious non--genomic viral DNA, oncogene or proto-oncogene, and can be incorporated into experimenter's karyomit(e) or can be episome or transgenosis DNA.The detection of whole viral genome or whole infectious virus DNA is importantly related with infection.For example, mention the HSV origin cause of formation thing of herpes keratitis, its existence for complete infectious gene group in the energy showed cell is necessary, and then it can divide or fully generation in the middle part of cell that infects.

Biological sample directly obtains from experimenter or patient usually, and can consist of tissue, cell sample, or blood, CSF or synovia sample or other hydrobiology samples.The dna molecular or the fiber that are used for extension or molecular comb can extract from biological sample.Sample can obtain from that live or non--experimenter alive.The experimenter comprises the animal that is sensitive to virus disease, comprises people, non--the people Mammals, such as ox, and ox, sheep, goat, horse, pig, dog, cat and non--people primate; Bird, such as chicken, turkey, duck, goose, ostrich, emu or other birds; Reptilia, Amphibians and other animals.

Make dna molecular extension or that combed and the genome that is suitable for identifying infectivity or biologic activity being arranged, infectious virus, one or more probe contacts of oncogene or proto-oncogene DNA.For detecting dna virus, adopt one or more probes of Corticovirus genomic 80～100% or any middle subdomain or value, or preferred one group of probe.Dna virus can be single or double-stranded.Representational dna virus comprises simplexvirus, and such as hsv (HSV) 1 and 2 types, papilloma virus and hepatitis virus are such as hepatitis B virus.Also select in a similar manner and retrovirus, such as HIV, preferably be integrated into the probe of the retrovirus combination of host chromosome as provirus, and the preferred one group of probe that covers provirus genomic 80～100% that adopts.

Probe can corresponding to known infective virus strain genomic at least 5% and preferred 80～100%, maybe can be designed to comprise and know for specific and plant copying of virus, the combination of the gene of breeding or pathogenic necessity.

Described probe can be corresponding to HSV-1 (NC_001806.1) (SEQ ID NO:15), HSV-2 (NC_001798.1) (SEQ ID NO:16), HIV1 (NC_001802.1) (SEQ ID NO:17), HIV2 (NC_001722.1) (SEQ ID NO:18), HBV (NC_003977.1) (SEQ ID NO:19), HPV16 (NC_001526.2) (SEQ ID NO:20), HPV18 (X05015.1) (SEQ ID NO:21), HPV31 (J04353.1) (SEQ ID NO:22), HPV33 (M12732.1) (SEQ ID NO:23) and HPV45 (X74479.1) (SEQ ID NO:24), it is by reference their accession number merging also.

One or more probes or probe groups can comprise 2 kinds, the probe subgroup of 3 kinds or how different different marker marks of usefulness.Preferably, this probe is used for different piece or the section of configuration or identifying virus genome or infectious virus dna molecular or another pathogenicity bo dna molecular such as oncogene or the proto-oncogene of mensuration genome or infectious virus DNA.

In another embodiment, the present invention is directed to and detect or identify infectivity in mammalian cell, tissue or the biological fluid or the method for genomic viral polynucleotide sequence, it comprises existence or the amount of using molecular comb to detect infectious virus polynucleotide in cell, tissue or the biological fluid or genomic viral polynucleotide.An embodiment consists of the method for quantitative infectivity or genomic viral polynucleotide sequence again, and it comprises that using molecular comb to detect cell, tissue or biological fluid compares in the control sample that does not infect that different time points obtains or in addition the infectious virus polynucleotide in the similar sample or the amount of genomic viral polynucleotide.

Also be encompassed in the existence that detects virus in the biological sample or the method that detects virus replication, and the method for especially in mammalian cell, tissue or biological fluid, longitudinally following the tracks of the rearrangement of virus replication or viral genome or infectious virus DNA.The method can be by molecular comb from suitable sample or get the DNA that the sample of specific period obtains and carry out, and is used for that cell or tissue is hidden or the existence of the viral DNA of replication-competent virus DNA or permutatation.

The present invention also belongs to the effect of estimating anti--viral therapy with molecular comb to detect existing, arrange or measuring of from the sample that the experimenter obtains infectivity or genomic viral DNA, with anti--viral agent treatment experimenter or carry out other anti--viral therapies, and monitoring or revalue after using molecular comb technical finesse as herein described infectivity or the method for existence, arrangement or the amount of genomic viral DNA.

Implement the test kit of molecular comb, it contains molecular comb equipment and/or reagent, one or more probes of being combined with infectious polynucleotide, and randomly one or more cells, tissue or biological fluid sample and carry out molecular comb, DNA extend or other conventional ingredients of probe hybridization.In some embodiments, test kit can contain the one group of probe that detects or identify the genomic viral DNA in the dna molecular of being combed, its Corticovirus genome, infectious viral nuclei acid, at least 5% of oncogene or former-oncogene, especially 80,85,90,95,99 or 100%.The minimum length of probe or probe groups is 1kb.In some embodiments, test kit can also contain for detection of or the software of the infectious sequence of classifying, and in other embodiments, test kit can additionally comprise the operation instruction that detects viral genome or infectious viral nuclei acid for the method according to this invention.

[description of drawings]

Figure1 .HSV-1 specific probe that can be used according to the invention ExamplePoint diagram represents the comparison of HSV-1 genome sequence (transverse axis) and HSV-1 specific probe (Z-axis), with mutually positioning different probe.Some HSV-1 fragments (HSV-S54 ,-B52 and-S58) with the sequence hybridization of inverted repetition, thereby exist with 2 copies.The HSV-1 fragment that is listed as in the Table A can relatedly reach with different color and show, to detect the HSV-1 genome.In this example, following related 99% the HSV-1 genome that allows to cover of 41 kinds of probes (on available 63 kinds of probes, Table A).HSV-S54 ,-P4 ,-P5 ,-S4 and-the B4 fragment is corresponding to obvious H121kb probe.Significantly the H256kb probe by 19 kinds of overlapping probes (HSV-B7 ,-B8 ,-S14 ,-B10 ,-S16 ,-B13 ,-S18 ,-B15 ,-S21 ,-B19 ,-S23 ,-B21 ,-B22 ,-S30 ,-S31 ,-S32 ,-B26 ,-P8 and-the B28 fragment) form.45kb H3 probe is by HSV-B30 ,-S44 ,-S45 ,-B38 ,-B39 ,-B40 ,-S49 ,-B44 ,-B45 ,-B46 ,-B48 and-the S54 fragment forms.Because the existence of the sequence of inverted repetition, H4(13kb) and H6(6.5kb) probe by HSV-B52 and-the S58 fragment forms; The HSV-S59 fragment also is present in the H4 probe.Finally, (HSV-S60 ,-B58 reach-B60) form the H57.5kb probe to 3 overlapping fragmentses.Formation is specific to the position probe of label of the genomic given zone of HSV-1 for H1, and H3 and H5 probe are red (digoxigenin mark, black surrounds), and for H2, H4 and H6 probe are green (biotin labeling, grey frames).The size of position probe (being rounded to nearest kb) is in each respectively indication below the frame.

Fig. 2. extract the comparison of 2 kinds of methods of HSV-1DNA from virus particle.(A) example of the HSV-1DNA that on the surface of silanization, is combed.At comb with standard phenol chloroform (left image) or the extraction flow process of modifying from the right image of the HSV-1(of agarose connector-embeddings) after the HSV-1DNA solution of extraction, fiber shows with intercalator YOYO-1, and observes at camera-equipped epifluorescence microscope.Figure shows 2 images of the feature that is each extracting method.Scale is indicated with bar.(B) histogram is presented at the accumulated frequence of dna fiber size number within the given length interval of containing on the genomic dna of HSV-1 separate particles.Interval width is 10 μ m.Thus, for example, the 3rd representative measured number in ［ 30～40 μ m ］ interval.Left figure shows and uses standard phenol: the length of every HSV-1DNA fiber of record (654 measure) in the dna solution that the chloroform extraction method is extracted.The meta size of fiber is 18 μ m, i.e. 36kb.Right figure shows the length distribution (2322 measure) of the dna fiber that obtains with substituting method.In this case, the meta size of distribution is 42 μ m, i.e. 84kb.

Fig. 3. the detection of the genomic isomer of HSV-1 in the dna solution of the cell extraction of virus particle and infection.(A) to by the example of the FISH of the HSV-1DNA that combed.Indication corresponding to the schematically illustrating of the different possible tissue of the crossing pattern of the genomic different isomerization body of HSV-1 (the digoxigenin mark-H1, H3 and H5 probe represent with black surround; Vitamin H-mark-H2, H4 and H6 probe show with grey frame table).Also to indicate just fully more than the signal such as the minimum essential requirement crossing pattern in the definition of " analysis of the signal that HSV-1 detects " part.4 representational linear hybridization chain (whites: texas Red/Alexa594-fluorescence: H1, H3 and H5 that show genomic each the complete isomer of HSV-1; Black: green Alexa488-fluorescence: H2, H4 and H6).More than the image of correspondence, show schematically illustrating of each HSV-1 genome isomer.(B) at Vero, the histogram of the distribution genome isomer of HSV-1KOS strain in the virus particle that produces in COS7 and the Neuro2A clone.As be described in " embodiment " part selection and analysis hybridization signal.In this example, identify and classified each is tested total 405 hybridization signals.Each bar represents genomic each constitutional isomer of HSV-1.In this example, being distributed in the virus particle of COS7 cell considerably of HSV-1KOS strain isomer distributes, yet P and IS isomer are at isomer more frequently in the virus particle that Neuro2A and Vero clone produce.The molar distribution (χ-2 check) such as this distribution statistics afterwards is different from with learning.(C) histogram of the distribution of the genome isomer of HSV-1 strain Sc16 and KOS in the different cells (BSR, COS-7, Neuro2a and Vero cell) that infect.As be described in " embodiment " part selection and analysis hybridization signal.In these examples, selection and classification are from 405 signals of each generation.The molar distribution (χ-2 check) such as BSR, COS-7, the distribution statistics of the HSV-1 strain KOS that produces among the HSV-1 strain Sc16 that produces in Neuro2a and the Vero cell and the COS-7 are equivalent to with learning.In the cell of Neuro2a and Vero infection, P and IS ratios of the isomers IL and ILS isomer are more frequent.

Fig. 4. from the method for mouse and rabbit corneal extraction genomic dna.The example of the genomic dna of (A) on the surface of silanization, being combed.The flow process of describing in the embodiment part is after cornea extracts, and comb genomic dna solution reached before camera-equipped epifluorescence microscope is observed and shows with intercalator YOYO-1.Left image shows the representational image of the DNA that the quilt that extracts from the healthy mice cornea is combed, and right image is the representational image of the DNA that combs from the quilt that the Healthy Rabbits cornea extracts.Molecular comb carries out with low density, to allow to measure the length of genomic dna fiber.Scale is indicated with bar.(B) histogram shows the accumulated frequence of dna fiber size number within the given length interval of containing on the genomic dna of HSV separate particles.Interval width is 25kb.Thus, for example, measure number in the 5th representative ［ 225～250kb ］ interval.Left figure shows the length of every dna fiber of record (10069 measure) in the dna solution that the mouse cornea extracts.The meta size of fiber is 204kb.In this sample, about 31% genomic dna fiber presents the above size of 200kb.Right figure shows an example of the length distribution (8336 measure) of the dna fiber that obtains from rabbit corneal.In this case, the meta size of distribution is 196kb, and the above pars fibrosa of length 200kb is 27%.

Fig. 5. the histogram of the distribution of the genomic isomer of HSV-1 in the mouse cornea that infects.The mouse cornea that infects from HSV-1 extracts total DNA, comb and and HSV-1-specific probe hybridization.In this example, identify and total 18 hybridization signals of classifying.Each bar represents genomic each constitutional isomer of HSV-1.

The detection of the replicative intermediate of HSV-1 in the mouse cornea that Fig. 6 .HSV-1 infects.Figure shows the 2 examples (white signal: texas Red/Alexa594-fluorescence: H1, H3 and H5 corresponding to the compound HSV-1 genomic dna that copies concatemer; Black signal: green Alexa488-fluorescence: H2, H4 and H6).Epigraph is described the signal by at least 2 imaginary overlapping HSV-1 genome constitutions that are comprised of IS and P isomer, and hypograph shows the HSV-1 concatemer that is comprised of imaginary at least ILS and IL signal.

Fig. 7. the genomic detection of non-standard HSV-1 in the mouse cornea that HSV-1 infects.(A) example of the non-standard HSV-1 of the mouse cornea of self-infection.Figure shows the representational example (white signal: texas Red/Alexa594-fluorescence: H1, H3 and H5 of the crossing pattern that does not correspond to one of genomic standard isomer of HSV-1; Black signal: green Alexa488-fluorescence: H2, H4 and H6).The standard crossing pattern corresponding to the ILS isomer that the DNA extraction thing that the scale indication is combed at the quilt of the Vero cell of HSV-1 strain Sc16-infection obtains (H1 of vitamin H-mark, H2B and H3 probe are with grey frame and signal indication; The H2A of digoxigenin-mark and H5 be with white edge and signal indication, and the H4 of Alexa488-mark and H6 probe are with black surround and signal indication).This routine show tags is to estimate the HSV-1 specific probe H1～H6 of the ratio of non--norm structure in the H4/H6 district.(C) the non-standard H4/H6 on the HSV-1 strain Sc16 in the Vero cell extract.The 1st crossing pattern shows the Alexa488 fluorescent signal (black signal) corresponding to the various size of H4/H6 probe, with corresponding to fragment H1, the Alexa594 fluorescence (grey signal) of H2B or H3 probe, or corresponding to the AMCA/Alexa350 fluorescent signal (white signal) of the part of the H2A of maximum 10kb or H5 probe alternately.The 2nd example present the Alexa488 fluorescent signal (black signal) of various size and the AMCA/Alexa350 fluorescent signal (white signal) that centered on by Alexa594 fluorescent signal (grey signal) between alternately.The 3rd example shows the single repetition of the Alexa488 fluorescent signal (black signal) that is centered on by Alexa594 fluorescent signal (grey signal).The indication scale.(D) histogram of the distribution between standard and the non-standard structure in the H4/H6 district of HSV-1.As be described in 367 hybridization signals of " embodiment " part selection and analysis.In this example, 80% H4/H6 probe is corresponding to theoretical construct.

The detection of provirus HIV-1DNA in Fig. 8 .ACH-2 cell culture.All the histogram demonstration is with number of signals (0.644kb/ class) in the given length interval of the function of a FISH signal length or the size of space between 2 FISH signals (with kbp).Thus, for example, the 1st histogrammic the 2nd representative is with the number of measuring at ［ 7.86～8.50kb ］ interval.(A) example of the provirus HIV-1DNA that is positioned at karyomit(e) 7p15 of use G248P87988G9 and G248P86255A8F clay.Indication corresponding to the schematically illustrating of the tissue of the crossing pattern of the provirus HIV-1DNA that integrates (the digoxigenin mark-the HIV-1 probe is with black surround and signal indication; Vitamin H-mark-the F clay is with white edge and signal indication).Left and right histogram shows between the difference F green Alexa488-fluorescent signal of clay G248P87988G9 and the HIV-1 texas Red/Alexa594-fluorescent signal, and the distribution of the size of space between HIV-1 texas Red/Alexa594-fluorescent signal and the green Alexa488-fluorescent signal of F clay G248P86255A8.Middle histogram shows the distribution of HIV-1 texas Red/Alexa594-fluorescent signal size.(B) example of the normal allele at 7p15 seat.Indication is corresponding to the schematically illustrating of the tissue of the crossing pattern of normal allele (vitamin H-mark-F clay with grey frame and signal indication).Histogram shows the distribution of the size of space between the F green Alexa488-fluorescent signal of clay G248P87988G9 and the green Alexa488-fluorescent signal of G248P86255A8.(C) the provirus HIV-1DNA that separates.Indication is corresponding to the schematically illustrating of the tissue of the crossing pattern of the HIV-1 of the form of separating (digoxigenin mark-HIV-1 probe with black surround and signal indication).Histogram shows the distribution of HIV-1 texas Red/Alexa594-fluorescent signal size.(D) example of the provirus HIV-1DNA that is positioned at karyomit(e) 7p15 of use G248P84833H9F clay.Indication corresponding to the provirus HIV-1DNA(digoxigenin mark of integrating-the HIV-1 probe is with black surround and signal indication); And the schematically illustrating of the tissue of the crossing pattern of wild-type seat (vitamin H-mark-G248P84833H9F clay with white edge and signal indication).Histogram shows the distribution of HIV-1 texas Red/Alexa594-fluorescent signal size.

[detailed Description Of The Invention]

The present invention cause infectious virus polynucleotide in the sample or infectious virus come source DNA fast, specificity and responsive the detection, it avoids the obvious constraint of being forced such as ELISA by amplification method such as PCR or serology test.

Opposite with art methods, molecular comb technology of the present invention allows the detection of the infectious or strong poison success partly of complete virus genome or viral polynucleotide sequence, causes the diagnosis of the improvement of acute or latent viral infection.

Method of the present invention causes and cost effective pattern effective with the time, and without radioactive type and its genomic structure that detects reliably HSV of operation with retraining.And method of the present invention causes in antiviral agent treatment, considers to follow the tracks of after the treatment of any type no matter the existence of virus infection.

The method that the present invention relates to is for the genomic detection of genomic existence, especially HSV of the cell dna virus of the infection of vitro detection eukaryotic cells.Described method comprises the representational nucleic acid of given virus and is specific at least the probe of HSV DNA or the hybridization step of one group of probe.

Molecular comb technology that can be used according to the invention is by people such as Bensimon, US patent No.6, and 303,296 and by people such as Lebofsky, WO2008/028931 discloses, and disclosure merges by reference at this.The contriver recognizes, except traditional culture technique, the technology of there is no can detect infectious form virus complete or non--the genomic existence of permutatation, seek thus to use and applicable these technology, to detect infectious virus DNA.The method of comb is never as the effective tool test that detects this viral DNA form, but considers the hereditation of understanding this sequence in the specific genotypic environment, only for the position of the long sequence of the DNA that is inserted into cellular genome.Detect non-limiting being illustrated in down of infectious virus (HSV) DNA by the method by the modification of contriver exploitation.

Contriver's standard of having modified is extracted flow process, with from virus particle isolated viral genomic dna.Generally speaking, replace 0.1% sarcosyl in the normal process, 0.1%SDS is used for the cracking of low-melting agarose connector virus particle.Also development approach to extract genomic dna from cornea, is thought the existence of HSV DNA in the cornea that diagnostic purpose research infects.

The present invention relates to detect the viral DNA, especially the HSV DNA that in the nucleic acid of the cell, tissue or the biological fluid that infect, contain and the method for HIV DNA.The representational nucleic acid that described method comprises described viral DNA with cover whole described viral DNA and allow to identify the specific probe of the rearrangement within the representational nucleic acid of described viral DNA or the hybridization step of probe groups.

Term used herein " nucleic acid " and especially " the representational nucleic acid of viral DNA " are specified the upholder can attach to and extend to this paper definition, and one or more molecules of more special nucleic acid by using any type that the molecular comb technology extends; Nucleic acid molecule comprises especially genomic dna of DNA(, especially viral DNA, or cDNA) and RNA(mRNA especially).Nucleic acid molecule can be strand or two strands.

It is all or about the information of purpose necessity of the present invention, it is present on the described viral DNA that the described nucleic acid of " the representational nucleic acid of described viral DNA " expression contains genetic information.This term comprises genomic viral DNA, such as the viral DNA that is incorporated into host chromosome, and can produce and to lack the specific gene set of pieces but when especially in host cell, expressing, can be infective infectious virus, infectious virus DNA, reach the virogene to host cell performance pathogenicity bo effect, such as viral oncogene.

Former-oncogene comprises those normal genes, and it can change the oncogene that causes cell to grow or divide in unadjusted mode into when being changed by sudden change.Former-oncogene has various cell function, and some are provided for fissional signal, and other can play a role in apoptosis.The function and structure feature of oncogene, the nucleotide sequence that comprises them, for well known, and by reference by people such as J.Nicolas, Karger Publishers(2010), ISBN3805585764,9783805585767(244 page or leaf) Human Cancer Viruses:Principles of Transformation and Pathogenesis merges, and it merges by reference.

Oncogene comprise defective form former-oncogene.The oncogene of single copy can cause not controlled Growth of Cells.Representational oncogene comprises ras, myc, src, Her-2/neu, hTERT and Bcl-2.The function and structure feature of oncogene comprises their nucleotide sequence, for well known, and by reference by people such as L.M.Surhone, Betascript Publishers(2010), ISBN6130361599, the 9786130361594(172 page or leaf) Oncogene:Gene, Mutation, Tumor, Apoptosis, Gene Expression, Protein, Cell Growth, Cellular Differentiation merges.Oncogene further describe and identify by reference last access on http://www.cancerquest.org/index.cfm page=780(2011 April 11) and quote Cooper G.Oncogenes.Jones and Bartlett Publishers, 1995and Vogelstein B, Kinzler KW; The Genetic Basis of Human Cancer.McGraw-Hill:1998 merges, and the two merges by reference.Former-oncogene can comprise virus transduction or viral integrase, point mutation, insertion mutation, gene amplification, chromosome translocation and/or protein-protein interaction to the reactivation process of oncogene.Can induce the virus of former-oncogene activation to comprise HBV and HCV(hepatocellular carcinoma), the HTLV(leukemia), the HPV(uterine cervix, anus and penile cancer), the sarcoma of HSV-8(Ka Boxi), merkel's cells polyomavirus (merkel's cells cancer) reaches, the EBV(Burkitt lymphomas, Hodgkin lymphoma, post-transplant lymphoproliferative disorders and nasopharyngeal carcinoma).

In particular implementation, the nucleic acid samples that be used for to extend is genomic dna, the especially total genomic dna of the cell or tissue that infects or more preferably chromogene group DNA(nuclear DNA), and/or its fragment.Term " nucleic acid " is at the representational nucleic acid that refers in particular to one or several karyomit(e) and/or one or several chromosome segment used herein.Described fragment can be any size, and the longest molecule reaches several Mbp.Described fragment usually comprise 10 and 2000kb between, more preferably 20 and 500kb between, and average about 300kb.

The nucleic acid samples that uses in the method for the invention can obtain from biological fluid or from the tissue of biological origin, described sample or for example organize from the people, and non-human mammal or bird separate.

As defined herein, probe is the representational nucleic acid that has with the viral DNA of this paper definition, especially with the polynucleotide of the ability of RNA and DNA hybridization, and nucleic acid/polypeptide crossbred or polypeptide.This term comprises especially mRNA of RNA() and the especially viral cDNA of DNA(or virus genom DNA) molecule, peptide nucleic acid(PNA) (PNA), and protein structure domain.Described polynucleotide or nucleic acid hybrids comprise at least 100,300,500 Nucleotide usually, preferred at least 700,800 or 900 Nucleotide, and more preferably at least 1,2,3,4 or 5kb or by at least 100,300,500 Nucleotide, preferably at least 700,800 or 900 Nucleotide, and more preferably at least 1,2,3,4 or 5kb form.For example, can use 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15kb or greater than 15kb, especially 30,50,100 or the probe of 150kb.This probe or one group of probe can correspondence or Corticovirus genomic, especially infectious virus genomic 5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,98,99 or 100%.Nucleic acid probe can be the polynucleotide of list or double-stranded polynucleotide or modification.One group of continuous probe groups is understood specific part or the whole genome of covering gene group, or overlaps each other about this part.The probe that is fit to can be based on viral polynucleotide sequence, such as disclosed herein those, and can be identified or synthetic by any appropriate means.But probe radioactivity, fluorescence or other means mark or marks of being known by this area.

At least 6 Nucleotide of the common specific binding of polypeptide probe reach the more preferably sequence of at least 10,15 or 20 Nucleotide.As used herein, probe sequence when probe is polypeptide, is interpreted as the sequence of described polypeptid specificity combination.

" part " given zone, it means the continuous nucleotide of the sequence of described given zone at this paper.Part of the present invention can comprise at least 15 or 20 continuous nucleotides, and preferably at least 100,200,300,500 or 700 continuous nucleotides, and more preferably at least 1, the described given zone of 2,3,4 or 5 continuous kb or by at least 15 or 20 continuous nucleotides, preferably at least 100,200,300,500 or 700 continuous nucleotides, and more preferably at least 1,2, the described given zone of 3,4 or 5 continuous kb forms.For example, part can comprise 1,2,3,4,5,6,7,8, the described given zone of 9,10,11,12,13,14,15 continuous kb or by 1,2, the described given zone composition of 3,4,5,6,7,8,9,10,11,12,13,14,15 continuous kb.

In particular implementation, at least a in the probe of use or the probe of use is the nucleotide variants that the part with a chain of target nucleic acid shows the probe of 100% complementary sequence.The sequence of described variant can have at least 70,80,85,90 or 95% complementarity with the sequence of the part of a chain of target nucleic acid.Described variant can especially be different from probe, and it is 100% same or complementary by 1～20 in former nucleotide sequence, and preferably by 1～10, nucleotide deletion inserts and/or more preferably replaces, especially by, 1,2,3,4,5,6,7,8,9 or 10 nucleotide deletions insert and/or more preferably replacement.In particular implementation, variant keeps and nucleic acid target sequence, similarly or 100% ability that be complementary to probe hybridization, the especially specific hybrid (especially under the hybridization conditions of this paper definition) of the sequence of nucleic acid target same with 100%.

Term " complementary sequence " refers in sight of the present invention " complementation " and " oppositely " or " " sequence namely can be interacted and the DNA chain-ordering with DNA chain combination of described sequence by Watson-Crick the palindrome.

In particular implementation of the present invention, will be used for implementing probe of the present invention or one or more probes with one or more haptens (biological example element and digoxigenin) mark, and use for these haptenic specific antibodies and show.Can allow to detect rearrangement within the given viral DNA for the different haptenic use of given probe or probe groups.Described probe as defined herein mark reaches as is described in patent application WO2008/028931, and it merges by reference.

One group of probe used herein comprises at least 2 kinds of probes.For example, described probe groups can be comprised of 2～20 kinds of probes (2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 kind of probe).Number of probes in the group is no more than 10,20,30,40 usually, and 50,60,70,80,90 or 100 kind of probe, depend on the sensitivity and the probe length that need; One group of probe is preferably at most by 2,3,4,5,6,7,8,9 or 10 kind of probe form.

Probe of the present invention or probe groups not only allow to detect whole full genomic viral DNA, and identify the rearrangement that can occur within genomic viral DNA.For example, the probe groups that is used for the HSV detection of using among the embodiment 1 allows by centering on distinct zones U _LAnd U _SThe sequence of repetition of reversing between the detection of the different HSV genome isomer that produce of homologous recombination.And, for detection of each probe groups of HSV (H1～H6) by (being used for by H1 the digoxigenin of the fragment that H3 and H5 probe form with 2 kinds of different different haptens; Being used for by H2 the vitamin H of the fragment that H4 and H6 probe form) several probes (3～19 kinds of different fragments) of mark form.The haptens of one or more fragments allows to produce different color-fluorescence array by the change of other haptens (no matter this haptenic any character) within given probe groups, and it allows the rearrangement within this given zone of identifying virus DNA.

Molecular comb also can be the patient of the cancer sensitivity that is caused by virus infection for development, for example, is in those the useful instrument of early diagnosis of risk that former-oncogene changes to oncogene.Really, molecular comb can distinguish integration and the additive type viral genome.In the situation that viral DNA is integrated, by using the particular probe group, molecular comb can allow to measure the viral DNA of whether integrating contain complete virus oncogene or no its be integrated into cell former-oncogene is contiguous.

The present invention also can use in conjunction with gene therapy.By using the particular probe group to each transgenosis and/or virus vector, molecular comb is provided for assessing based on the effect of the gene therapy of virus vector and the strong instrument of security.Really, molecular comb can distinguish integration and the additive type transgenosis.Integrate in the situation of (carrier of slow virus and retrovirus base) in transgenosis, by using the particular probe group, molecular comb can allow to measure that the viral DNA of whether integrating is integrated into gene (insertion mutagenesis) or cell is former-and oncogene is contiguous.In the situation of episome (adenovirus carrier, AAV or hsv vector) in transgenosis; Molecular comb can be for quantitatively transgenosis is useful in the cell mass of growth, but and auxiliary definition be used for the orthochronous that transgenosis is used again.

Particular implementation of the present invention comprises following methods and product.Method for detection of the infectious virus polynucleotide in the biological sample, it comprises: from described sample separation, extraction or obtain in addition polynucleotide, the described polynucleotide of molecular comb are to form the polynucleotide that extend, make the polynucleotide of described extension contact the hybridization of the sample that detection probes and quilt are combed with one or more probes of the infectious polynucleotide sequence of identification.This method can be to carry out from the tissue of experimenter's acquisition or the biological sample of cell sample by use, for example, and blood, blood plasma, serum, CSF, synovia sample or some other kinds biological fluids from the experimenter.The polynucleotide that use in this method can extract from the tissue that obtains from the experimenter or cell sample or from the component of biological fluid.Generally speaking, sample can obtain from the experimenter who lives, but sample also can obtain from the experimenter of death.Sample can be from people or other Mammalss such as ox, ox, sheep, goat, horse, pig, dog, cat and non--people primate, or from other animals such as the fowl species such as chicken, turkey, duck, goose, ostrich, emu or other birds obtain.Method can be carried out with polynucleotide, and described polynucleotide are DNA, and it also can contain or comprise infectivity or non--infectious gene papova DNA or non--infectious non--genomic viral polynucleotide such as DNA or RNA.Method polynucleotide that detect or that analyze can be incorporated into experimenter's DNA or can be the additive type form thus.Polynucleotide to be detected can with in conjunction with dna virus, comprise that one or more probes of strand and double-stranded DNA virus contact.For example, polynucleotide can with in conjunction with simplexvirus, such as one or more probes contact of hsv (HSV).Spendable other probes are viral such as papilloma virus in conjunction with other, hepatitis B virus, or retrovirus such as HIV.In some embodiments, method can be used in conjunction with at least 80%, 90%, 95% or 99% genome of virus or one group of probe of infectious DNA.In other embodiments, one or more probes that use in the method can comprise at least 2 group probes with different marker marks, for example, to allow to identify the virus genomic different piece of different probe combination or the rearrangement in section or the permission identifying virus genome.

Other particular implementation of the present invention comprise:

Detect, identify or visual mammalian cell, tissue or biological fluid in infectivity or the method for genomic viral polynucleotide sequence, it comprises existence or the amount of using molecular comb to detect infectious virus polynucleotide in cell, tissue or the biological fluid or genomic viral polynucleotide.

The quantitative method of infectivity or genomic viral polynucleotide sequence, it comprises that using molecular comb to detect cell, tissue or biological fluid compares at different time points or from the control sample that does not infect that not homology or clinical sample obtain or in addition the infectious virus polynucleotide in the similar sample or the amount of genomic viral polynucleotide.

Detect or follow the tracks of virus in the mammalian cell and exist or copy or method that viral genome is reset, it comprise for hide in cell, tissue or the biological fluid or copy in viral DNA or the existence of the viral DNA of permutatation implement molecular comb.

Estimate the method for the effect of anti--viral therapy, comprise existence, arrangement or the amount of using molecular comb to detect from the sample that the experimenter obtains infectivity or genomic viral DNA, treat described experimenter with anti--viral agent, and use molecular comb to revalue existence, arrangement or the amount of infectious virus among the described experimenter or genomic viral DNA.

Cover 80～100% HSV, HIV, the genomic one group of probe of HBV or HPV.

Be used for implementing the test kit of molecular comb, it comprises: molecular comb equipment and/or reagent in conjunction with one or more probes of infectious polynucleotide, reach randomly one or more cells, tissue or biological fluid sample.The test kit of the one group of probe of the genomic viral DNA in the dna molecular that inclusion test or evaluation are combed.Test kit also comprises for detection of or the software of the infectious sequence of classifying.

The present invention also relates to following biomaterial, it is deposited in La Photographie microbial preservation center (Collection Nationale De Cultures De Microorganismes on April 20th, 2010 according to budapest treaty, CNCM), Institut Pasteur25Rue du Docteur Roux, 75724Paris Cedex15:HSV-B4(CNCM I-4298), HSV-B19(CNCM I-4299), HSV-Sc54(CNCM I-4300), reach HSV-P4(CNCM I-4301).Each biomaterial contains the polynucleotide passage of the probe that detects corresponding to HSV.Therefore the present invention relates to each these polynucleotide passage and relates to their any combination.

[embodiment]

[embodiment 1: hsv detects]

[from the preparation of the DNA connector of the embedding of virus particle]

From virus particle by standard phenol: chloroform extraction method (Ben-Zeev, Weinberg et al.1974) or extract HSV-1DNA by the process (Lebofsky, Heilig et al.2006) of the modification that is described in the people such as Lebofsky, the two merges by reference.In brief, with the HSV-1 particle with 5 * 10 ⁶The concentration of virus particle/mL is resuspended to 1 * PBS, and with 1:1 than with the 1.2%w/v that in PBS, prepares low-fusing point agarose (Nusieve GTG, ref.50081, Cambrex) solution, thoroughly mix in 50 ℃.The virus particle of 90 μ L/agarose mixture dropped into connector-formation hole (BioRad, ref.170-3713) and remain in 4 ℃ cool off at least 30min.Make the virus particle of embedding at 0.1%SDS-0.5M EDTA(pH8.0) in the solution in 50 ℃ of cracking 30 minutes.At 0.5M EDTA(pH8.0) in the buffer reagent after the step of room temperature washing in 10 minutes 3 times, with connector by the incubation that spends the night in 50 ℃ with 2mg/mL Proteinase K (Eurobio code GEXPRK01, France) digestion in 250 μ L digestion buffer reagents (0.5M EDTA, pH8.0).It is very productive replacing sarcosyl to use 0.1%SDS, and allows the viral DNA of very high-quality extraction to be collected.

[preparation of the DNA connector of the embedding of the cell of self-infection]

The extraction of the HSV-1DNA of the cell culture of self-infection (BSR, COS-7, Neuro2A and Vero) is carried out as describing before (Schurra and Bensimon2009).In brief, by the cell with 5000g precipitation infection in centrifugal 5 minutes, with 2 * 10 ⁶The concentration of cell/mL is resuspension in 1 * PBS buffer reagent, and with 1:1 than with in 1 * PBS, prepare low-the 1.2%w/v solution of fusing point agarose (Nusieve GTG, ref.50081, Cambrex) thoroughly mixes in 50 ℃.The cell of 90 μ L/agarose mixture is dropped into connector-formation hole (BioRad, ref.170-3713), and remain in 4 ℃ and cool off at least 30min.

Lysis in the piece is carried out (Schurra and Bensimon2009) as describing before.In brief, agarose plugs in 50 ℃ at the 0.5M EDTA(pH8 of 250 μ L), 1% sarcosyl, be incubated overnight in 250 μ g/mL Proteinase K (Eurobio, code:GEXPRK01, the France) solution, then at Tris10mM, in the EDTA1mM solution in twice of room temperature washing 30min.

[preparation of the DNA connector of the embedding of the cornea of self-infection]

Infecting the mouse cornea of collecting HSV-1 strain Sc16 infection latter stage, and remaining on

In (Eurobio code EYEMAX00, France) substratum.Be room temperature during 15 minutes with after 1 * PBS solution rinsing 3 times, whole cornea is cut into small pieces.At GIBCO ^TMThe 0.3mg/mL Collagenase A type (Roche, coding 10103578001) of preparation in 1 * Hanks ' Balanced salts solution HBSS buffer reagent (Invitrogen, France, coding 14060040), and 0.8mg/mL GIBCO ^TMOrganize cracking to reach 16h in 37 ℃ in the Dispase (Invitrogen, France, coding 17105-041).Lysate is by precipitating with 5000g in centrifugal 10 minutes, in 1 * PBS buffer reagent with 1 * 10 ⁶～2 * 10 ⁶The concentration resuspension of cell/mL, and with 1:1 than with 1 * PBS in prepare low-the 1.2%w/v solution of fusing point agarose (Nusieve GTG, ref.50081, Cambrex) thoroughly mixes in 50 ℃.The cell of 90 μ L/agarose mixture drops into connector-formation hole (BioRad, ref.170～3713), and remains in 4 ℃ and cool off at least 30min.

Lysis in the piece is carried out (Schurra and Bensimon2009) as describing before.In brief, agarose plugs in 50 ℃ at the 0.5M EDTA(pH8 of 250 μ L), 1% sarcosyl, be incubated overnight in 250 μ g/mL Proteinase K (Eurobio, code:GEXPRK01, the France) solution, then at Tris10mM, in the EDTA1mM solution in twice of room temperature washing 30min.

[DNA finally extracts and molecular comb]

Processing is used for as describing before comb DNA(Schurra and Bensimon2009 from the connector of the DNA of the embedding of virus particle or cornea).In brief, will plug in 68 ℃ at MES0.5M(pH5.5) melt 20min in the solution, and add β-gelase (New England Biolabs, ref. M0392S, MA, USA) of 1.5U and keep incubations to reach 16h in 42 ℃.Then dna solution drops into the teflon storage and carries out molecular comb by use molecular comb system (Genomic Vision S.A., Paris, France) and molecular comb cover glass (20mm * 20mm, GenomicVision S.A., Paris, France).Make the surface of being combed in 60 ℃ of dryings 4 hours.

[the synthetic and mark of HSV-1 probe]

To list in Table A with respect to the coordinate of whole probes of Genbank sequence NC_001806.1.Probe size is striden 1110～9325bp in this example.

The HSV-1 specific probe passes through from CNRS(Prof.Marc Labetoulle, laboratoire de virology mol é culaire et structurale, UMR CNRS2472-INRA1157, Gif-sur-Yvette, the SacI of the HSV-1sc16 strain that France) obtains or BspEI(be New England Biolabs Inc. respectively, Beverly, MA, USA coding R0156L and R0156L) enzymatic digestion or by using LR Taq archaeal dna polymerase (Roche, test kit code: 11681842001) use the primer of listing in table B and using from the DNA of HSV-1sc16 to produce as the long-range PCR of template DNA.SacI and BspEI HSV-1 fragment are connected respectively pNEB193 plasmid (New England Biolabs Inc., Beverly, MA, USA, coding N3051S) into SacI and XmaI-digestion.The PCR product is used

TA clone's test kit (Invitrogen, France, encoded K 455040) connects to advance

Carrier.For the checking purpose, checked order in the two ends of each probe.Obvious H1(21kb), H2(56kb), H3(45kb), H4(13kb), H5(7.5kb) and H6(6.5kb) probe is the mixture of listing in several adjacent or overlapping probe of Table A.Visual for the non-standard structure that involves H4 and H6 probe, significantly the H2 probe is split into 2 probe H2A(31kb) and H2B(25kb).

By using the conventional mark that flow process is carried out probe that causes at random.For vitamin H-11-dCTP mark, use according to manufacturer's operation instruction

DNA test kit (Invitrogen, code: 18094-011, CA, USA) is except the permission labeled reactant spends the night.For 11-digoxigenin-dUTP and Alexa488-7-OBEA-dCTP, will be from the dNTP of test kit mixture with showing specific mixture replacement among the C.Each plasmid mark in independent reaction with 200ng.For the isomer classification, with H1, H3, H5 probe 11-digoxigenin-dUTP mark, and with H2, H4 and H6 probe vitamin H-11-dCTP mark.Visual for the non-standard structure that involves H4 and H6 probe, with H1, H2B and H3 probe be with vitamin H-11-dCTP mark, with H2A and H5 probe with 11-digoxigenin-dUTP mark, with H4, H6 Alexa488-7-OBEA-dCTP mark.Reaction product is visual on sepharose, with synthesizing of conclusive evidence DNA.

[by hybridization and the detection of HSV-1 probe on the viral DNA of combing]

Also basically as being described in before Schurra and Bensimon, 2009(Schurra and Bensimon2009) carry out later step, it merges by reference.In brief, with the mixture of the probe of mark (each probe of 250ng, consider that probe details synthetic and mark see lower) and 10 μ g Pacific herring sperm DNAs and 2,5 μ g people Cot-1DNA (Invitrogen, ref.15279-011, CA, USA) ethanol precipitates together, is resuspended to hybridization buffer (50% methane amide, the 2 * SSC of 20 μ L, 0.5%SDS, 0.5% sarcosyl, 10mM NaCl, 30%Block-aid(Invitrogen, ref.B-10710, CA, USA)).Probe solution and probe is upper in 90 ℃ of heat-sex change 5min together at hybridization instrument (Dako, ref.S2451), and hybridization is spent the night in 37 ℃ of maintenances on hybridization instrument.With slide glass at 50% methane amide, among 2 * SSC the washing 3 times, and in 2 * SSC solution in room temperature washing 5min through 3 times.Detect antibody layer and in Block-Aid their corresponding dilution be described in table D and E.For each layer, add the antibody-solutions of 20 μ L to slide glass, and use the cover glass of being combed to cover, and with slide glass in wet atmosphere in 37 ℃ of incubation 20min.With slide glass between each layer and in the end after the layer at 2 * SSC, in the 1%Tween20 solution in room temperature washing 3min through 3 times.Be the isomer classification, for H1, H3 and H5 probe, use the anti-digoxigenin of mouse (the Jackson Immunoresearch of the texas Red coupling of 1:25 dilution, France) antibody, and for H2, H4 and H6 probe, use the Streptavidin antibody (Invitrogen, France) of the Alexa488-coupling of 1:25 dilution to detect as first antibody.As the 2nd layer, the goat anti-mouse (Invitrogen, France) of the Alexa594-coupling of use 1:25 dilution and the anti-Streptavidin of biotinylated goat (Vector Laboratories, UK) of 1:50 dilution.For the H2 that increases, the Alexa488-fluorescent signal of H4 and H6 probe, other detection layers by use identical 1:25 dilution be used for the 1st layer the Alexa488 coupling-Streptavidin realizes.For involve H4 and H6 probe non--norm structure visual, for H2A and H5 probe, use little mouse-anti-digoxigenin (Jackson Immunoresearch of the AMCA-coupling of 1:25 dilution, France) antibody, for H1, H2B and H3, the Streptavidin antibody (Invitrogen of the Alexa594-coupling of use 1:25 dilution, French), and the anti-Alexa488 of rabbit of 1:25 dilution detects as first antibody.As the 2nd layer, use the goat anti-mouse (Invitrogen of the Alexa594-coupling of 1:25 dilution, France), the biotinylated goat of 1:50 dilution anti--goat of the Alexa488-coupling of Streptavidin (Vector Laboratories, UK) and 1:25 dilution resists-mouse.For the H1 that increases, the AMCA/Alexa350 signal of the Alexa594-fluorescent signal of H2B and H3 probe and H2A and H5 probe, other detection layers by use respectively identical 1:25 dilution be used for the 1st layer the Alexa594 coupling-the goat Chinese People's Anti-Japanese Military and Political College mouse of the Alexa350 coupling of Streptavidin and 1:25 dilution realizes.In the end after the washing step, all the glass cover slide dewaters and dry air in ethanol.

[analysis of the signal that HSV-1 detects]

Be the direct visual HSV-1 fiber of being combed, cover glass is with the Prolong (Invitrogen of 20 μ L, France ref P36930)-YOYO-1 iodide (Molecular Probes, coding Y3601) mixture (1/1000v/v) sealing and with epifluorescence microscope (the ImageXpress Micro of the inverted automatization of equipping 40 * object lens, Molecular Devices, USA) scanning.Measure YOYO-1-dyeing dna fiber length and use the extension factor of 2kb/ μ m, with in house software GVlab04.2.1(Genomic Vision S.A., Paris, France) change kb(Schurra and Bensimon2009 into).

Be the isomer classification, need not any sealing substratum use equip epifluorescence microscope (the ImageXpress Micro of the inverted automatization of 40 * object lens, Molecular Devices, USA) scanning from the hybridization of virus particle or cornea prepared product-DNA that combed, and signal can be by in house software (Gvlab0.4.2) vision or automatically detect.Consideration is by for H1, H3, and the texas Red of H5/Alexa594-fluorescence and for H2, the FISH signal that the continuous signal of the Alexa488-fluorescence of H4 and H6 forms, and the signal that is formed by the continuous signal corresponding to one of pattern described below:

(a) texas Red of minimum 28kb length/Alexa594-fluorescent signal directly is corresponding to the Alexa488-fluorescent signal of H4 and another texas Red of minimum 3kb length/Alexa594-fluorescent signal afterwards.

(b) texas Red of minimum 28kb length/Alexa594-fluorescent signal directly is corresponding to the Alexa488-fluorescent signal of H6 and another texas Red of minimum 3kb length/Alexa594-fluorescent signal afterwards.

(c) minimum 3kb Alexa488 fluorescent signal directly is the texas Red/Alexa594 fluorescent signal corresponding to H1 afterwards, and the next-door neighbour is corresponding to the Alexa488-fluorescent signal of H4 and another texas Red of minimum 3kb length/Alexa594-fluorescent signal.

(d) minimum 3kb Alexa488-fluorescent signal, directly be the texas Red/Alexa594-fluorescent signal corresponding to H1 afterwards, the next-door neighbour is corresponding to the Alexa488-fluorescent signal of H6 and another texas Red of minimum 3kb length/Alexa594-fluorescent signal.

Then the FISH signal of selecting depends on the pattern classification of the continuous FISH signal of analysis:

The HSV-1(Hayward that the probe array that is comprised of the H1/H2/H3/H4/H5/H6 probe or pattern (a) are categorized as prototype (P) form, Jacob et al.1975).

Pattern H1/H2/H3/H6/H5/H4 or pattern (b) are categorized as the HSV-1(Hayward of reverse weak point (IS) genome district form, Jacob et al.1975).

Pattern H3/H2/H1/H4/H5/H6 or pattern (c) are categorized as long (IL) genome district (Hayward, Jacob et al.1975) of the palindrome of HSV-1.

Pattern H3/H2/H1/H6/H5/H4 or pattern (d) are categorized as the HSV-1(Hayward of long and short (ILS) genome of palindrome district form, Jacob et al.1975).

The distribution of being observed by χ side's verification test significantly is different from the hypothesis (for the event that 4 kinds of isomer are quite counted, (Bataille and Epstein1997)) of the distribution of expection, and when the p-value of observing in 0.05 acceptance when following.

Visual for the non-standard structure that involves H4 and H6 probe, with the hybridization of the cellular preparations of self-infection-DNA that combed need not any sealing substratum, use epifluorescence microscope (the ImageXpress Micro of the inverted automatization of equipment 40 * object lens, Molecular Devices, USA) scanning, and signal can be in the upper vision-based detection of in house software (Gvlab0.4.2).Whole signals that selection is comprised of the continuous signal for the Alexa488-fluorescence of H4 and H6.To be categorized as norm structure corresponding to the signal of one of following pattern:

(e) continuous signal that is formed by the Alexa594-fluorescent signal of minimum 3kb, the Alexa488-fluorescent signal corresponding to the 13kb of H4 probe afterwards, corresponding to the AMCA/Alexa350-fluorescent signal of the 7.5kb of H5 probe with corresponding to the Alexa488-fluorescence of the 7kb of H6 probe.

(f) continuous signal that is formed by the Alexa594-fluorescent signal of minimum 3kb, the Alexa488-fluorescent signal corresponding to the 7kb of H6 probe afterwards, corresponding to the AMCA/Alexa350-fluorescent signal of the 7.5kb of H5 probe with corresponding to the Alexa488-fluorescence of the 13kb of H4 probe.

All other signals are categorized as the non-standard structure.The ratio that compares standard and non-standard structure.

[from the extraction of the HSV-1DNA of virus particle]

During sample preparation, shear many dna moleculars at random site, because not controlled operating physical force causes the high variability of the DNA size for preparing.Show that high-molecular-weight DNA can use the glass cover slide to extend by molecular comb, when its in agarose connector of fusing during deproteinated (Lebofsky and Bensimon2003).Thus, the dna molecular of analysis is variable length, average about 300kb, and the longest molecule reaches several Mbp.

Because HSV-1DNA never is used for molecular comb, the contriver has at first estimated by 2 kinds of different methods: standard phenol: chloroform extraction and by (Lebofsky, Heilig et al.2006) describe as the quality about the dna fiber of length that is described in that method that " embodiment " part modifies a little extracts.

As be shown in Fig. 2, detect without the fiber more than the size 80 μ m.This size (152kb) with the genomic greatest expected of HSV-1 is consistent, and it considers the constant elongation factor of 2kb.With two kinds of methods, we do not detect only 152kb length dna fiber, because because inevitably power operation still has random dna to shear, and advance nucleocapsid (Vlazny, Kwong et al.1982) because compare the shorter dna molecular of the total length standard HSV-1 viral genome shell that can become.

But when using standard phenol: when chloroform method was extracted, the meta size of dna fiber was 36kb, and 1.2% Fiber Phase is longer than 140kb.On the contrary, when the substituting flow process that realizes using us from the DNA extraction of the virus particle of agarose connector-embedding the time, the meta size of HSV-1DNA fiber is 84kb, 2.5% Fiber Phase is longer than 140kb.Although the ratio of long molecule is low, the dna molecular that has the quilt of many length to comb can be used for analyzing, because the fiber that has several ten thousand quilts to comb at the glass cover slide.

The standard method that allows by the molecular comb analysis is compared in these result's indications, has been improved the quality of the DNA that combs from the quilt of virus particle extraction by the substituting method of contriver's exploitation.

[the genomic structure of HSV-1 and its distribution in the virus particle]

The contriver uses molecular comb with the HSV-1DNA of even extension from the cell extraction of virus particle and infection, and makes HSV-1DNA that the quilt that obtains combs and adjacent and overlapping HSV-1-specificity DNA probing needle hybridization (Fig. 1 of mark; H1, H3, H5: red texas Red/Alexa594-fluorescence; H2, H4, H6: green Alexa488-fluorescence), to measure the genomic structure of HSV-1 (Fig. 3 A).Immunofluorescence microscopy inspection (Fig. 3 B) has presented for from COS7,405 polychrome linear models of each productive rate of the HSV-1KOS strain virus particle that Vero and Neuro2A clone produce, and it satisfies evaluation criteria (seeing " embodiment " part).The signal classification display, being distributed in the virus particle of COS7 cell considerably of HSV-1KOS strain isomer distributes, however P and IS isomer are being isomer more frequently in the virus particle that Neuro2A and Vero clone produce.

The cell that the HSV-1 that the quilt of hybridization is combed infects, the relatively distribution of 4 kinds of isomer between the Sc16 that in different clones (BSR, COS7, Neuro2A and Vero), produces and the KOS HSV-1 strain.Classification is corresponding to each generation and satisfy the 405 polychrome linear models (seeing " experimentation " part) of evaluation criteria.Suitable (χ2-test,chi-square test) (at BSR, Vero is in Neuro2a and COS-7 cell) in statistics ground in the whole generations that are distributed in HSV-1Sc16 between 4 kinds of isomer.In an identical manner, find that distribution is suitable for the HSV-1 strain KOS that produces in the COS-7 cell.Observably, first, the contriver has found that IS and P isomer are the HSV-1DNA strain KOS prepared products from the principal mode of Vero and Neuro2A cell, and the IL isomer is the isomer of less existence.

The contriver has found that molecular comb technology as herein described is the strong method of its distribution in the structure of individual molecule horizontal analysis HSV-1 genomic dna and quantitative biology sample.

[from the extraction of the genomic dna of mouse and rabbit corneal]

Molecular comb comprises that with the cell of self-separation the dna solution of cultured cells (that is, the cell strain of foundation, Immortalized primary cell) or biological fluid (that is, peripheral blood lymphocyte, amnion cell) successfully carries out (Gad, Klinger et al.2002; C aburet, Conti et al.2005).But, people's cornea is the solid tissue that has by following 5 layers of composite structure that forms: corneal epithelium, be rich in the BowmanShi film of collagen, matter between the cornea that is formed by the collegen filament along the corneal cell rule of the interconnection that sparsely distributes-arrange, acellular property DescemetShi film reaches, corneal endothelium.In order to extract genomic dna from cornea, the contriver has developed the ad hoc approach of isolated cornea cell before carrying out with standard procedure.Test comprises the mechanical disintegration of cornea and the different methods of enzymatic digestion.The latter draws optimum, and optimizes by the dissimilar proteolytic enzyme (that is, trypsinase, Collagenase A, Dispase) that uses different concns.Fig. 4 A and B show with the mouse of 0.3mg/ml Collagenase A and 0.8mg/ml Dispase digestion 16h and the result's that rabbit corneal obtains a example.Extract as for the standard that is used for molecular comb, at random site split gene group DNA.Thus, for two types cornea, the genomic dna molecule of analysis is variable length, has average about 200kb, and the longest molecule is at 1Mb(Mb) more than.The dna fiber size of extracting from cornea is slightly less than the typical sizes that can obtain from the cell that separates (on average about 300kb, the longest molecule reaches several Mbp).

[detection that HSV-1 infects in the mouse cornea]

Therefore the contriver is suitable for and has used molecular comb to the DNA that the mouse cornea that infects from HSV-1 extracts, and reaches and above-mentioned HSV-1 specific probe hybridization.As being shown in Fig. 5, this causes the isomer of the genomic all types of HSV-1 in the cornea that detects mouse infection.

Except detecting ripe HSV-1 genome, molecular comb process of the present invention allows to detect concatemer (Fig. 6), and indicator virus copies in the cornea of the sample of analyzing actively.

[detection of non-in the cell of infection and the mouse cornea-canonical form]

The contriver detected HSV-1 genomic non--norm structure (Fig. 7 A), it is probably being occurred by restructuring during the virus replication in the mouse cornea extract that infects and the cell extract that infecting.

Adjacent and the overlapping HSV-1 specific probe of mark is at DNA extraction thing hybridization (Fig. 7 B of the Vero cell that infects from HSV-1 strain Sc16 of being combed; H1, H2A, H3: red Alexa594-fluorescence presents with grey; H2B, H5: blue AMCA/Alexa350-fluorescence presents with white; H4, H6: green Alexa488-fluorescence presents with black), to estimate the ratio of non--norm structure in the H4/H6 district.Total 367 polychrome linear models of classifying 20%(73) find to have non--standard H4/H6 structure.

Molecular comb causes the visual of non-standard structure, reaches the unlimited combination by their possible bar code, is the strong method of analyzing them.

Table A

Table B:

Be used for the primer sequence by long-range PCR synthesising probing needle.Will be from the extract of the DNA of HSV-1 strain Sc16 as template.

Table C:

Replace the mixture of the dNTP mixture use that causes at random test kit, it is used for dig-dUTP and Alexa488-7-OBEA-dCTP mark.The concentration of indication is the final concentration in the labeled reactant.The dNTP mixture that provides is provided adds together the dNTP of non--mark and the dNTP of mark, it is intended to vitamin H-11-dCTP-mark.

Table D:

Antibody and other haptens-binding molecule tabulation for detection of probe.

Table E:

Be used for by 2 or 3 layers of fluorescent detection probe form.Dilution to each detection agent is indicated with parantheses.Abbreviation reference table D.

[embodiment 2: the human immunodeficiency virus detects]

[from the preparation of the DNA connector of the embedding of ACH-2 cell cultures]

Operation instruction according to the author is cultivated ACH-2 clone (Clouse, Powell et al.1989).As be described in (Schurra and Bensimon2009) and extract DNA.In brief, cell is with 10 ⁷The concentration of cell/mL is resuspended to 1 * PBS, with 1:1 than with 1 * PBS in prepare low-the 1.2%w/v solution of fusing point agarose (Nusieve GTG, ref.50081, Cambrex) thoroughly mixes in 50 ℃.The cell of 90 μ L/agarose mixture drops into connector-formation hole (BioRad, ref.170～3713), and remains in 4 ℃ and cool off at least 30min.Agarose plugs in 50 ℃ at the 0.5M EDTA(pH8 of 250 μ L), 1% sarcosyl, 250 μ g/mL Proteinase K (Eurobio, code:GEXPRK01, France) be incubated overnight in the solution, then at Tris10mM, in the EDTA1mM solution in twice of room temperature washing 30min.

[final extraction and the molecular comb of DNA]

Processing is used for as describing before comb DNA(Schurra and Bensimon2009 from the connector of the DNA of the embedding of ACH-2 cell).In brief, will plug in 68 ℃ at MES0.5M(pH5.5) melt 20min in the solution, and add β-gelase (New England Biolabs, ref.M0392S, MA, USA) of 1.5U and keep incubations to reach 16h in 42 ℃.Then dna solution drops into the teflon storage and carries out molecular comb by use molecular comb system (Genomic Vision S.A., Paris, France) and molecular comb cover glass (20mm * 20mm, Genomic Vision S.A., Paris, France).Make the surface of being combed in 60 ℃ of dryings 4 hours.

[the synthetic and mark of HIV-1 probe]

To list in table F with respect to the coordinate of 3 kinds of probes of Genbank sequence M19921.1.Stride 2927～3749bp in this routine middle probe size.

The HIV specific probe uses LR Taq archaeal dna polymerase by long-range PCR, and (Roche, the test kit code: 11681842001), use is listed in the primer of table G and is produced as template DNA from the DNA of HIV pNL4-3.The PCR product uses TA clone's test kit (Invitrogen, France, encoded K 455040) connects to advance

Carrier.For the checking purpose, checked order in the two ends of each probe.

2 of the insertion point of side joint HIV-1 kinds of F clay G248P87988G9 and G248P86255A8 or comprise a kind of F clay G248P84833H9(Ishida of HIV-1 provirus insertion point in the ACH-2 cell, Hamano et al.2006), according to Human Mar.2006Assembly(NCBI Build36.1), and with the HIV-1 probe by using routine to cause at random the flow process mark.For vitamin H-11-dCTP mark, use according to manufacturer's operation instruction

DNA test kit (Invitrogen, code: 18094-011, CA, USA) is except the permission labeled reactant spends the night.For digoxigenin-11-dUTP, will be from the dNTP of test kit mixture with showing specific mixture replacement among the C.Each plasmid of mark 200ng/F clay in independent reaction.Detect for whole HIV-1, with HIV-1 digoxigenin-11-dUTP mark, and with F clay vitamin H-11-dCTP mark.Reaction product is visual on sepharose, with synthesizing of conclusive evidence DNA.

[by hybridization and the detection of HIV-1 probe on the viral DNA of combing]

Also basically as before be described in Schurra and Bensimon, 2009(Schurra and Bensimon2009) carry out later step.In brief, with mixture and 10 μ g Pacific herring sperm DNAs and 2, the 5 μ g people Cot-1DNA(Invitrogen of the probe (each probe of 250ng) of mark, ref.15279-011, CA, USA) ethanol precipitation together, be resuspended to hybridization buffer (50% methane amide, 2 * SSC, the 0.5%SDS of 20 μ L, 0.5% sarcosyl, 10mM NaCl, 30%Block-aid(Invitrogen, ref.B-10710, CA, USA)).Probe solution and probe is upper in 90 ℃ of heat-sex change 5min together at hybridization instrument (Dako, ref.S2451), and hybridization is spent the night in 37 ℃ of maintenances on hybridization instrument.With slide glass at 50% methane amide, among 2 * SSC the washing 3 times, and in 2 * SSC solution in room temperature washing 5min through 3 times.Detect antibody layer and in Block-Aid their corresponding dilution be described in table D and E.For each layer, add the antibody-solutions of 20 μ L to slide glass, and use the cover glass of being combed to cover, and with slide glass in wet atmosphere in 37 ℃ of incubation 20min.With slide glass between each layer and in the end after the layer at 2 * SSC, in the 1%Tween20 solution in room temperature washing 3min through 3 times.

For the HIV-1 probe, use little mouse-anti-digoxigenin (Jackson Immunoresearch of the texas Red coupling of 1:25 dilution, France) antibody, and for the F clay, use the Streptavidin antibody (Invitrogen, France) of the Alexa488-coupling of 1:25 dilution to carry out the detection of whole HIV-1 as first antibody.As the 2nd layer, the goat anti-mouse (Invitrogen, France) of the Alexa594-coupling of use 1:25 dilution and the anti-Streptavidin of biotinylated goat (Vector Laboratories, UK) of 1:50 dilution.For the Alexa488-fluorescent signal of the F clay that increases, other detection layers by use identical be used for the 1st layer the 1:25 dilution the Alexa488 coupling-Streptavidin realizes.Washing step in the end, all the glass cover slide dewaters and dry air in ethanol.

[analysis of the signal that HIV-1 detects]

Will from the hybridization of ACH-2 cellular preparations-DNA that combed, need not any sealing substratum, use epifluorescence microscope (the ImageXpress Micro of the inverted automatization of equipment 40 * object lens, Molecular Devices, USA) scanning, and signal can be by in house software (Gvlab0.4.2) vision or is automatically detected:

● use 2 kinds of F clay G248P87988G9 and the G248P86255A8 of the insertion point of side joint HIV-1, use the elongation factor of 2kb/ μ m to consider and measure FISH signal (Schurra and Bensimon2009) corresponding to one of following pattern:

■ FISH signal is comprised of the continuous signal for the texas Red of HIV-1/Alexa594-fluorescence.Measure the whole signal of the corresponding HIV proviral DNA that separates

■ FISH signal array is by for the signal chains by the texas Red of the HIV-1 of 2 interval side joints/Alexa594-fluorescence, and forms corresponding to 2 continuous signals of the Alexa488-fluorescence of F clay sequence.The gap length of measuring whole texas Red/Alexa594 fluorescent signal and this signal of side joint reaches corresponding to be integrated into the 7th chromosomal HIV-1 proviral DNA (Ishida, Hamano et al.2006) at 7p15.

■ has the FISH signal array of 2 Alexa488-fluorescent signals by being spaced apart.Carry out the measurement corresponding to the gap length at the 7p15 seat of integrating without the HIV-1 proviral DNA.

● use F clay G248P84833H9 to comprise HIV-1 provirus insertion point, use the elongation factor of 2kb/ μ m to consider and measure FISH signal (Schurra and Bensimon2009) corresponding to one of following pattern:

■ FISH signal is comprised of the continuous signal for the texas Red of HIV-1/Alexa594-fluorescence.Measure the whole signal of the corresponding HIV proviral DNA that separates

■ FISH signal array is comprised of the continuous signal chain for the texas Red of HIV-1/Alexa594-fluorescence, and it is by 2 Alexa488-fluorescent signal side joints corresponding to F clay sequence.Measuring whole texas Red/Alexa594 fluorescent signal reaches corresponding to be integrated into the 7th chromosomal HIV-1 proviral DNA (Ishida, Hamano et al.2006) at 7p15.

■ has the FISH signal of a long Alexa488-fluorescent signal corresponding to the 7p15 seat of integrating without the HIV-1 proviral DNA.

[detection of HIV-1 in the ACH-2 cell culture]

The contriver has used molecular comb, in ACH-2 clone, to detect the provirus (Clouse that complete HIV-1 integrates, Powell et al.1989), it is in its genome, NT5C3(cytosol 5'-NT III on 7p14.3) contains the HIV-1(Ishida of unique form of integrating in the gene, Hamano et al.2006).The F clay of the mark of side joint insertion point (G248P87988G9 and G248P86255A8) is compared the HIV-1 probe of mark, is being hybridized simultaneously on the ACH-2DNA that combs.From by the 124HIV-1FISH signal of one or both F clay side joints measure 10.2kb+/-mean sizes of 0.8kb, size (9 corresponding to the expection of HIV-1,7kb) (Fig. 8 A, HIV probe: red texas Red/Alexa594-fluorescence, F clay: green Alexa488-fluorescence).Detect the normal allele of NT5C3 gene, and the measurement of the gap length between F clay G248P87988G9 and the G248P86255A8FISH signal causes the mean sizes of 32.8 ± 1.8kb, the size that is better than gently the expection of 31kb is according to Human Mar.2006Assembly(NCBI Build36.1).This result shows that molecular comb can be with the further information (Fig. 8 B) of serving relevant this seat by its resolving power.In addition, in the ACH-2DNA prepared product that this is combed, measure the HIV-1FISH signal of 133 separation, mean sizes be 10.05kb+/-0.79kb(Fig. 8 C).This with unique HIV-1 insertion point of the expection of describing before in pairs than (Ishida, Hamano et al.2006), and prompting its be present in the ACH-2 genome, in another or other insertion point of HIV-1, or the HIV-1 of nonconformable form continues in ACH-2 nuclear.When HIV-1 probe that the F of the mark that comprises insertion point clay (G248P84833H9) is compared mark when being hybridized simultaneously on the ACH-2DNA that combs, carry out similar observation (Fig. 8 D).From 57HIV-1FISH signal within F clay signal measure 10.4kb+/-mean sizes of 0.5kb, size (9 corresponding to the proviral expection of HIV-1,7kb) (HIV probe: red texas Red/Alexa594-fluorescence, F clay: green Alexa488-fluorescence).In addition, also in the ACH-2DNA prepared product that this is combed, detect and measure the HIV-1FISH signal of 35 separation, mean sizes be 10.03kb+/-0.82kb(do not show).

These result's indications, molecular comb are to reach structure and quantitative its strong method of integrating of analyzing the HIV genomic dna in any biological sample in the individual molecule level in genomic dna.

Table F:

Title	Initial	Finish	Size (bp)
				HIV-S1	1	3026	3026
HIV-S2	3018	5944	2927
				HIV-S3	5961	9709	3749

The coordinate of 3 kinds of probes that use in this example is with respect to Genbank sequence M19921.1

Table G:

Primer sequence is used for by long-range PCR synthesising probing needle.To be used as template from the DNA extraction thing of HIV-1pNL-4.

[embodiment 3: detect oncogene by molecular comb]

To be similar to the mode that detects the HSV genomic dna in embodiment 1, probe design is for detecting the existence of viral oncogene.Probe design is and activated purpose viral oncogene 80～100% complementations, and carries out molecular comb.The result indicates the existence of activated oncogene among the experimenter, causes diagnosis and therapeutic intervention.

[embodiment 4: use molecular comb to detect the rearrangement of infectious virus DNA]

To be similar to the mode that detects the HSV genomic dna in embodiment 1, probe design is for detecting the existence of the different infectious virus DNA that arrange.Be designed to and activated purpose viral oncogene 80～100% complementations with the different hapten-marked different probe group of being identified by different painted fluorescent probes, and carry out molecular comb.The variation of the arrangement of virogene is used for the risk of the progress of diagnosis or the prognosis virus disease related with virus genomic rearrangement or disease in experimenter's cell, tissue or the biological fluid, such as by former-oncogene to the risk of the tumorigenesis character of the transformation of oncogene or induce.

[embodiment 5: by molecular comb monitoring genetic therapy]

To be similar to the mode that detects the HIV-1 of provirus form in embodiment 2, probe especially is designed to and the therapeutic adenovirus carrier of integrating or its transgenic sequence that carries 80～100% complementations.By using specially designed probe to carry out molecular comb, be integrated into the existing of transgenosis material of host chromosome with detection, and whether it is arranged with the form of express transgenic actively.Longitudinally follow the tracks of genetically modified amount among the experimenter, and determine when the administering therapeutic sexual gland virus carrier with whether again.

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[quoting adding]

Each document of quoting in the disclosure or censuring, patent, patent application or patent disclosure are by reference with its whole merging, especially about centering on the particular topic of quoting of reference in the text.But, disapprove any this reference formation background field, and keep the tolerance range of challenging the document of quoting and the right of suitably spending.

Claims (17)

1. for detection of the method for the infectious virus polynucleotide in the biological sample, it comprises:

From described sample separation, extraction or obtain in addition polynucleotide,

The polynucleotide that the described polynucleotide of molecular comb extend with formation,

The polynucleotide of described extension are contacted with one or more probes of the infectious polynucleotide sequence of identification,

The hybridization of the sample that detection probes and quilt are combed; Detect thus the infectious virus polynucleotide.

2. the process of claim 1 wherein that described biological sample is tissue sample, cell, serum, blood, CSF or the synovia sample that obtains from people, non--people Mammals or bird.

3. the method for claim 1 or claim 2, wherein said polynucleotide are the infectious or non--infectious virus DNA from the tissue of people, non--people Mammals or bird, cell, serum, blood, CSF or synovia sample extraction.

4. each method of claim 1～3, wherein said polynucleotide are integrated into DNA or episome or the biological fluid of people or non--people Mammals or bird.

5. each method of claim 1～4, wherein said polynucleotide one or more probe in detecting of being combined with the viral DNA of two DNA chain viruses.

6. each method of claim 1～5, wherein said polynucleotide with and simplexvirus, papilloma virus, one or more probe in detecting of the DNA combination of hepatitis virus or retrovirus.

7. each method of claim 1～6, wherein said one or more probes comprise one group of probe of being combined with the genomic dna of at least 1kb of virus.

8. each method of claim 1～7, wherein said one or more probes comprise at least 2 groups with the probe of the different marker marks of the fragment of selecting to be used for to allow the genomic different piece of identifying virus or section or permutatation.

9. the method for claim 8, wherein said group probe covers 5～100% HSV or HIV genome.

10. detect or identify infectivity in mammalian cell, tissue or the biological fluid or the method for genomic viral polynucleotide sequence, it comprises existence or the amount of using molecular comb to detect infectious virus polynucleotide in cell, tissue or the biological fluid or genomic viral polynucleotide.

11. the quantitative method of infectivity or genomic viral polynucleotide sequence, it comprises that using molecular comb to detect cell, tissue or biological fluid compares in the control sample that does not infect that different time points obtains or in addition the infectious virus polynucleotide in the similar sample or the amount of genomic viral polynucleotide.

12. detect or follow the tracks of the method that viral existence in the mammalian cell or virus replication or viral genome are reset, it comprise for hide in cell, tissue or the biological fluid or copy in viral DNA or the existence of the viral DNA of permutatation implement molecular comb.

13. estimate the method for the effect of anti--viral therapy, it comprises:

Use molecular comb to detect existence, arrangement or the amount of from the sample that the experimenter obtains infectivity or genomic viral DNA,

Treat described experimenter with anti--viral agent, and

Use molecular comb to revalue existence, arrangement or the amount of infectious virus among the described experimenter or genomic viral DNA.

14. the method for each of claim 9～12, it comprises the step of the method that defines in each that implement claim 1～8.

15. one group of probe, it covers 5～100% HSV or HIV genome.

16. be used for implementing the test kit of molecular comb, it comprises:

Molecular comb equipment and/or reagent,

For detection of or identify one or more probes of being combined with the infectious virus polynucleotide of the genomic viral DNA in the dna molecular combed, and

Randomly one or more cells, tissue or biological fluid sample, and

Randomly for detection of or the software of the infectious sequence of classifying.

17. biomaterial HSV-B4(CNCM I-4298), HSV-B19(CNCM I-4299), HSV-Sc54(CNCM I-4300), or HSV-P4(CNCM I-4301).

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